B system does not interfere with phage adsorption to . Strains either containing ( red ) or lacking ( black ) BREX system were infected with phage Φ3T then chloroform ‐ treated 15 min following infection . culture was plated on Φ3T ‐ sensitive B . subtilis plaques , representing extracellular , unadsorbed phages , were counted .

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B The system does not interfere with phage adsorption to bacterial cells. Strains either containing (red) or lacking (black) the BREX system were infected with phage Φ3T and then chloroform‐treated 15 min following infection. The culture was plated on Φ3T‐sensitive B. subtilis cells and plaques, representing extracellular, unadsorbed phages, were counted.


C Phage replication does not occur in BREX ‐ containing ( red ) , but is observed in BREX ‐ lacking ( black ) . Y ‐ axis represents relative phage concentrations normalized to value at beginning of infection , as measured by Illumina sequencing .

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C Phage DNA replication does not occur in BREX‐containing cells (red), but is observed in the BREX‐lacking strain (black). Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection, as measured by Illumina sequencing.


C , D Culture dynamics of non ‐ infected ( C ) or phage ‐ infected ( D ) cultures . Curves depict culture dynamics of strains lacking BREX ( black ) BREX ‐ containing ( red ) strains of B . subtilis BEST7003 , as well as a BREX ‐ containing where pglX methylase was deleted ( green ) . Axes error bars are as in Fig .

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C, D Culture dynamics of non‐infected (C) or phage‐infected (D) cultures. Curves depict culture dynamics of strains lacking BREX (black) and BREX‐containing (red) strains of B. subtilis BEST7003, as well as a BREX‐containing strain where the pglX methylase was deleted (green). Axes and error bars are as in Fig .


E Southern blot of phage Φ3T genome during infection . Numbers indicate time ( in min ) following infection ; U , uninfected . Probe was designed to match positions 94 , 645 - 95 , 416 in phage genome . Each lane contains 200 ng total .

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E Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645-95,416 in the phage genome. Each lane contains 200 ng total DNA.


( B - D ) of normalized bouton number ( ( B ) , total number of boutons / muscle surface ( μm2 × 1 , 000 ) ) , normalized axon ( C ) normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * Data information : In ( B - D bars are labelled with number of replicates .

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(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction. (n.s. = not significant; p<0.05 = *; p<0.001= ***; p<0.0001=**** Data information: In (B-D bars are labelled with the number of replicates.


( B , C ) Western Blot showing enrichments upon pulldown of same probes as in ( A ) incubated with increasing concentrations of purified recombinant GST - Ythdf ( B ) His - NT - Fmr1 ( C ) . of m6A / A signal is shown below blot as median + / - of triplicates . Both bind more efficiently upon methylation .

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(B, C) Western Blot showing the protein enrichments upon pulldown of the same probes as in (A) incubated with increasing concentrations of purified recombinant GST-Ythdf (B) and His-NT-Fmr1 proteins (C). Quantification of the m6A/A signal intensity is shown below the blot as median +/- SEM of the triplicates. Both proteins bind more efficiently upon methylation.


( D ) Results of polarization ( FP ) using Cy5 labelled probes containing four AAACU m6A consensus sites , with or without methylation , incubated with GST - Ythdf His - NT - Fmr1 . relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8 . Binding constants ( kd values ) were determined by fitting a Michaelis - Menten non - linear regression onto relative FP values in GraphPad Prism 8 , if applicable . respective binding constants are given in table next to graphs .

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(D) Results of fluorescence polarization (FP) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites, with or without the methylation, incubated with GST-Ythdf and His-NT-Fmr1. The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8. Binding constants (kd values) were determined by fitting a Michaelis-Menten non-linear regression onto the relative FP values in GraphPad Prism 8, if applicable. The respective binding constants are given in the table next to the graphs.


( B - D ) of normalized bouton number ( ( B ) , total number of boutons / muscle surface ( μm2 × 1 , 000 ) ) , normalized axon ( C ) normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * ) . Data information : In ( B - D , , bars are labelled with number of replicates .

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(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.05 = *; p<0.01 = **; p<0.001= ***; p<0.0001=****). Data information: In (B-D, , bars are labelled with the number of replicates.


( B ) Co - immunoprecipitation experiment in S2R + co - expressing FlagMyc - tagged Ythdf Myc - tagged Fmr1 . Ythdf was used as a bait via its Flag tag . lysate was treated with Benzonase as indicated to remove interactions enabled by .

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(B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA.


Pulldown of biotinylated probes of ( A ) repetitive GGACU sequences incubated with 4 pmol His - NT - Fmr1 increasing concentrations of GST - Ythdf indicated P - values were determined by unpaired , two - Student ' s t - .

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Pulldown of biotinylated RNA probes of (A) repetitive GGACU sequences incubated with 4 pmol His-NT-Fmr1 and increasing concentrations of GST-Ythdf The indicated P-values were determined by unpaired, two-tailed Student's t-test.


( A ) Representative Western blot of extracts of late pupae heads ( 85 - 95h ) from control , Ythdf # 0 / Ythdf # 0 Fmr1 ∆ 50 / Fmr1 ∆ 50 flies . membranes were probed with anti - Profilin or anti - Actin antibodies . ( B ) of Profilin ( mean ± SD ) obtained from three independent - extraction Western blot as in ( A ) . was performed using Fiji . p < 0 . 005 = * * ; p < 0 . 05 = * , measured with unpaired t - .

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(A) Representative Western blot analysis of protein extracts of late pupae heads (85-95h) from control, Ythdf#0/ Ythdf#0 and Fmr1∆50/Fmr1∆50 flies. The membranes were probed with anti-Profilin or anti-Actin antibodies. (B) Quantification of Profilin protein levels (mean ± SD) obtained from three independent protein-extraction and Western blot analysis as in (A). Quantification was performed using Fiji. p<0.005 = **; p<0.05 = *, measured with unpaired t-test.


( D ) of futsch mRNA obtained from isolated 3rd instar larvae brains of indicated genotypes via real - time PCR . Bars show average ± SD of biological triplicates . Bars are labelled with number of replicates .

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(D) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real-time PCR analysis. Bars show average ± SD of biological triplicates. Bars are labelled with the number of replicates.


( F ) of normalized bouton number ( total number of boutons / muscle surface ( μm2 × 1 , 000 ) ) of NMJ 6 / 7 in hemisegment A3 of indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 01 = * * ; p < 0 . 0001 = * * * * ) . Bars are labelled with number of replicates .

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(F) Quantification of normalized bouton number (total number of boutons/muscle surface area (μm2 × 1,000)) of NMJ 6/7 in hemisegment A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.01 = **; p<0.0001=****). Bars are labelled with the number of replicates.


E - G ) Representative Western blot of level ( E ) of translation efficiency of S2R + transfected with wildtype or mutated futsch reporter constructs upon control or Mettl3 KD ( F ) or Ythdf KD ( G ) . LacZ dsRNA was used for control KD . translation efficiency was calculated as of relative GFP level GFP mRNA level . mCherry served as a normalization control for both mRNA level . Bars show mean ± s . e . m of biological triplicates . P values were determined with a Student ' s t - . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ) .

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E-G) Representative Western blot analysis of the protein level (E) and quantification of the translation efficiency of S2R+ cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD (F) or Ythdf KD (G). LacZ dsRNA was used for the control KD. The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level. mCherry served as a normalization control for both the protein and mRNA level. Bars show mean ± s.e.m of biological triplicates. P values were determined with a Student's t-test. (n.s. = not significant; p<0.05 = *; p<0.01 = **).


A Transcriptome re - of ulp2Δ strains compared with WT ( MHY1379 ) from previous - seq experiments Two - dimensional agglomerative hierarchical clustering shows 2 , 903 significantly up - or down - regulated genes ( P < 0 . 05 ) in triplicate samples . Red blue indicate up down regulation of genes , respectively , their represents relative gene expression changes . B Summery of transcriptome data in ( A ) . gray colors indicate up - down - regulated genes , respectively , in ulp2Δ . pie graph shows percentages of significantly changed genes , except for 130 ribosomal genes ( RPs ) , are classified by FPKM ( Fragments Per Kilobase of Million reads mapped ) values in bar graph .

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A Transcriptome re-analysis of ulp2Δ strains compared with WT (MHY1379) from previous RNA-seq experiments Two-dimensional agglomerative hierarchical clustering shows 2,903 significantly up- or down-regulated genes (P < 0.05) in triplicate RNA samples. Red and blue indicate up and down regulation of genes, respectively, and their intensity represents the relative gene expression changes. B Summery of transcriptome data in (A). White and gray colors indicate up- and down-regulated genes, respectively, in ulp2Δ cells. The pie graph shows the percentages of significantly changed genes, except for 130 ribosomal protein genes (RPs), and are classified by FPKM (Fragments Per Kilobase of Million reads mapped) values in the bar graph.


C qRT - PCR of highly transcribed PMA1 , ADH1 PYK1 genes in ulp2Δ . Expression was measured relative to WT data were normalized to SPT15 expression . Error bars indicate standard deviations ( SDs ) from three independent preparations . FPKMs of each gene in WT are shown in bottom graph .

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C qRT-PCR analysis of the highly transcribed PMA1, ADH1 and PYK1 genes in ulp2Δ cells. Expression was measured relative to WT cells and data were normalized to SPT15 expression. Error bars indicate the standard deviations (SDs) from three independent RNA preparations. FPKMs of each gene in WT are shown in the bottom graph.


E ChIP using IgG - Sepharose or anti - Flag - agarose beads in strains expressing TAP - tagged Paf1 , Ubc9 , or Ulp1 or Flag - tagged Ulp2 . An untagged ( MHY500 ) was used as a negative control for immunoprecipitation of Ulp2 - Flag qPCR signals of indicated genes were quantitated normalized to an internal background control input . primer pairs used are indicated in ( D ) . presented as fold over background ; a value of 1 indicates no signal detected above background signal at a nontranscribed locus , as marked with horizontal . Error bars indicate SDs calculated from three independent preparations . Data information : Asterisks indicate statistically significant differences of Paf1 - TAP Ubc9 - TAP with Ulp1 - TAP Ulp2 - Flag with No tag in ( E ) using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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E ChIP analysis using IgG-Sepharose or anti-Flag-agarose beads in strains expressing TAP-tagged Paf1, Ubc9, or Ulp1 or Flag-tagged Ulp2. An untagged strain (MHY500) was used as a negative control for immunoprecipitation of Ulp2-Flag The qPCR signals of the indicated genes were quantitated and normalized to an internal background control and the input DNA. The primer pairs used are indicated in (D). Quantification presented as fold over background; a value of 1 indicates no signal detected above background signal at a nontranscribed locus, as marked with the horizontal line. Error bars indicate SDs calculated from three independent chromatin preparations. Data information: Asterisks indicate statistically significant differences of Paf1-TAP and Ubc9-TAP with Ulp1-TAP and Ulp2-Flag with No tag in (E) using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


F Occupancy of Rpb3 Ulp2 at CUP1 gene was determined by ChIP in a expressing Flag - tagged Ulp2 using anti - Rpb3 antibody anti - Flag agarose beads Mock ' indicates use of G beads without added antibody . For CUP1 induction , were harvested at indicated time points after adding CuSO4 . " Pro " " ORF " represent positions of PCR fragments 1 2 from CUP1 gene Black bar indicates a value of 1 , background signal . Error bars , SD from four independent experiments . Data information : Asterisks indicate significant differences between uninduced induced in ( F ) using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 )

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F Occupancy of Rpb3 and Ulp2 at CUP1 gene was determined by ChIP in a strain expressing Flag-tagged Ulp2 using anti-Rpb3 antibody and anti-Flag agarose beads Mock' indicates use of Protein G beads without added antibody. For CUP1 induction, cells were harvested at the indicated time points after adding CuSO4. "Pro" and "ORF" represent the positions of PCR fragments 1 and 2 from the CUP1 gene Black bar indicates a value of 1, the background signal. Error bars, SD from four independent experiments. Data information: Asterisks indicate significant differences between uninduced and induced cells in (F) using a two-tailed Student's t test (*, P <0.05; **, P <0.01)


G A graph of association of Ulp2 - Flag with genes classified by FPKM RPs ND indicate ribosomal genes genes not detected genes in our earlier - seq experiments , respectively . numbers above graph indicate total number of yeast genes in each expression category .

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G A percentage graph of association of Ulp2-Flag with genes classified by FPKM RPs and ND indicate ribosomal protein genes and genes not detected genes in our earlier RNA-seq experiments, respectively. The numbers above the graph indicate the total number of yeast genes in each expression category.


H Average plot of Ulp2 - Flag occupancy at transcribed genes . values of y - axis indicate rescaled fold - enrichment , setting maximum occupancy to 1 minimum occupancy to 0 , x - axis indicates normalized from transcription start sites ( TSS ) transcription end sites ( TES ) . dotted lines indicate TSS TES . ChIP - seq data were obtained from duplicate experiments .

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H Average plot of Ulp2-Flag occupancy at transcribed genes. The values of the y-axis indicate rescaled fold-enrichment, setting the maximum occupancy to 1 and the minimum occupancy to 0, and the x-axis indicates normalized distance from transcription start sites (TSS) and transcription end sites (TES). The dotted lines indicate the TSS and TES. The ChIP-seq data were obtained from duplicate experiments.


A Sensitivity of indicated mutants to 6 - AU . strains carried a URA3 plasmid , pRS316 , were spotted on SD - Ura with or without 6 - AU ( 100 μg / ml ) ; plates were incubated for 2 - 4 days at 30 ° C . ULP2 ulp2 ( C624A ) represent ulp2Δ : : HIS3 containing either pRS314 - ULP2 - FLAG or pRS314 - ulp2 ( C624A ) - FLAG .

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A Sensitivity of the indicated mutants to 6-AU. All strains carried a URA3 plasmid, pRS316, and were spotted on SD-Ura with or without 6-AU (100 μg/ml); plates were incubated for 2-4 days at 30°C. ULP2 and ulp2(C624A) represent ulp2Δ::HIS3 cells containing either pRS314-ULP2-FLAG or pRS314-ulp2(C624A)-FLAG.


ChIP analyses using anti - Rpb3 ( B ) antibodies in ulp2Δ Error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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ChIP analyses using anti-Rpb3 (B) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


ChIP analyses using anti - SUMO ( C ) antibodies in ulp2Δ Error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

ChIP analyses using anti-SUMO (C) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


D qRT - PCR of CUP1 mRNA in ulp2Δ . Data were normalized to ACT1 mRNA . CUP1 gene induction was performed Error bars represent SD from three samples . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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D qRT-PCR analysis of CUP1 mRNA levels in ulp2Δ cells. Data were normalized to ACT1 mRNA levels. CUP1 gene induction was performed Error bars represent the SD from three RNA samples. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


ChIP assays using anti - Rpb3 antibodies in ulp2Δ Pro " denotes # 1 PCR product of PMA1 , ADH1 PYK1 genes , while " ORF " indicates # 3 products of PMA1 # 2 of ADH1 PYK1 ; these are used in ensuing figures except where specified . Error bars indicate SD from four ( E ) independent assays . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 .

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ChIP assays using anti-Rpb3 antibodies in ulp2Δ cells Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes , while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from four (E) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01


ChIP assays using anti - SUMO ( F ) antibodies in ulp2Δ ; Pro " denotes # 1 PCR product of PMA1 , ADH1 PYK1 genes while " ORF " indicates # 3 products of PMA1 # 2 of ADH1 PYK1 ; these are used in ensuing figures except where specified . Error bars indicate SD from three ( F ) independent assays . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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ChIP assays using anti-SUMO (F) antibodies in ulp2Δ cells ;Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from three (F) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


A Sensitivity to 6 - AU of indicated expressing chromosomally integrated ULP2 - Myc derivatives . Assays were performed schematic diagram below shows full - Ulp2 with a 9Myc epitope tag segments defining N - C - terminal domains catalytic ULP domain ( UD ) .

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A Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD).


B Immunoblot of Ulp2 - Myc derivatives expressed in

B Immunoblot assay of the Ulp2-Myc derivatives expressed in the strains


C ChIP of Flag - Ulp2 derivatives performed with same PCR probes W303a carrying pRS424 - GAL1 plasmids that expressed N - terminally Flag - tagged Ulp2 derivatives - specifically , full - , NTD ( 1 - 403 ) CTD ( 667 - 1034 ) - were grown to mid - log phase in SD - Trp medium containing 2 % galactose . Error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences compared with WT , full of Ulp2 ( * , P < 0 . 05 ) .

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C ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives-specifically, full-length, NTD (1-403) and CTD (667-1034)-were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT, full length of Ulp2 (*, P <0.05).


D Co - IP for interaction between Ulp2 - Myc derivatives Flag - H2B in strains transformed with a pRS314 plasmid expressing Flag - tagged H2B . Immunoprecipitated ( IP ) from anti - Myc agarose beads were analyzed by immunoblotting ( IB ) using anti - Myc or anti - Flag antibodies . upper middle panels show anti - Myc blotting anti - Flag blotting , respectively . of bound H2B were measured relative to INPUT H2B shown in lower panel .

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D Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting (IB) using anti-Myc or anti-Flag antibodies. The upper and middle panels show anti-Myc blotting and anti-Flag blotting, respectively. The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel.


E association assays using strains After fractionation into ( pellet ) soluble ( supernatant ) components , fractions were analyzed by immunoblotting using indicated antibodies .

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E Chromatin association assays using strains After fractionation into chromatin (pellet) and soluble (supernatant) components, fractions were analyzed by immunoblotting using the indicated antibodies.


F Quantitation of association of Ulp2 - Myc derivatives in ( E ) . of Ulp2 in each fraction were measured by ImageJ then each signal was divided by total values for each Ulp2 derivative .

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F Quantitation of chromatin association of Ulp2-Myc derivatives in (E). The levels of Ulp2 protein in each fraction were measured by ImageJ and then each signal was divided by the total values for each Ulp2 derivative.


A Immunoblot of immunoprecipitated Flag - tagged histone H2B with anti - Flag ( bottom panel ) or anti - SUMO ( upper panel ) antibodies in WT , ulp2Δ , SUMO - KR or ulp2Δ SUMO - KR strains carrying empty vector or pRS426 - FLAG - HTB1 . One , two or three asterisks represent mono , di polysumoylated histones , respectively . Note that apparent diSUMO - H2B species ( * * ) in ulp2Δ SUMO - KR migrates more slowly than H2B linked to a SUMO dimer , suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines ( lane 8 ; also see weaker band in lane 6 ) .

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A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B with anti-Flag (bottom panel) or anti-SUMO (upper panel) antibodies in WT, ulp2Δ, SUMO-all KR or ulp2Δ SUMO-all KR strains carrying empty vector or pRS426-FLAG-HTB1. One, two or three asterisks represent mono, di and polysumoylated histones, respectively. Note that the apparent diSUMO-H2B species (**) in ulp2Δ SUMO-all KR cells migrates more slowly than H2B linked to a SUMO dimer, suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines (lane 8; also see weaker band in lane 6).


B Yeast strains of indicated genotypes carrying a YCplac33 - ULP2 cover plasmid were streaked on SD + 5 - FOA to evict YCplac33 - ULP2 ; were grown at 30 ° C for 3 days .

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B Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.


C Mutant htb1 - K123R ulp2Δ htb1 - K123R ULP2 strains with a pRS316 - ULP2 cover plasmid were grown on indicated plates at 30 ° C for 2 - 3 days . Three double mutant isolates were tested in parallel .

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C Mutant htb1-K123R ulp2Δ and htb1-K123R ULP2 strains with a pRS316-ULP2 cover plasmid were grown on the indicated plates at 30°C for 2-3 days. Three double mutant isolates were tested in parallel.


D Mutant ulp2Δ bre1Δ strains carrying a pRS426 - ULP2 ( URA3 ) cover plasmid indicated plasmids were grown on SD - Ura SD + 5 - FOA at 30 ° C for 2 - 3 days .

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D Mutant ulp2Δ bre1Δ strains carrying a pRS426-ULP2 (URA3) cover plasmid and the indicated plasmids were grown on SD-Ura and SD+5-FOA at 30°C for 2-3 days.


E Yeast strains of indicated genotypes carrying a YCplac33 - ULP2 cover plasmid were streaked on SD + 5 - FOA to evict YCplac33 - ULP2 ; were grown at 30 ° C for 3 days .

E Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.


A Immunoblot of immunoprecipitated Flag - tagged histone H2B using anti - Flag or anti - SUMO antibodies in indicated mutants , upper lower panels show Flag - H2B - SUMO Flag - H2B - ub , respectively . One , two or three asterisks indicate mono - , di - , poly - sumoylated histones ( upper panel ) , respectively , an arrowhead bullet point represent mono - ubiquitylated H2B , a non - specific band , respectively .

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A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B using anti-Flag or anti-SUMO antibodies in the indicated mutants, The upper and lower panels show Flag-H2B-SUMO and Flag-H2B-ub, respectively. One, two or three asterisks indicate mono-, di-, and poly-sumoylated histones (upper panel), respectively, and an arrowhead and bullet point represent mono-ubiquitylated H2B, and a non-specific band, respectively.


B Immunoblot of sumoylated in extracts prepared from strains Anti - Flag blotting for Flag - H2B was used to verify similar loading .

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B Immunoblot assay of sumoylated proteins in extracts prepared from the strains Anti-Flag blotting for Flag-H2B was used to verify the similar loading.


C double immunoprecipitation ( ChDIP ) of Flag - H2B - ub in indicated strains expressing Flag - H2B . first ChIP was performed with anti - Flag agarose , eluted samples were immunoprecipitated with an antibody against ubiquitin . PCR signals from indicated genes were normalized to an internal control input . error bars indicate SD from three independent samples . Data information : Asterisks indicate statistically significant differences compared with WT in * , P < 0 . 05 ; * * , P < 0 .

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C Chromatin double immunoprecipitation (ChDIP) of Flag-H2B-ub in the indicated strains expressing Flag-H2B. The first ChIP was performed with anti-Flag agarose, and the eluted samples were immunoprecipitated with an antibody against ubiquitin. PCR signals from the indicated genes were normalized to an internal control and the input DNA. The error bars indicate the SD from three independent chromatin samples. Data information: Asterisks indicate statistically significant differences compared with WT in *, P <0.05; **, P <0.01


D ChDIP of Flag - H2B - SUMO in indicated strains expressing both Flag - H2B HA - SUMO . samples were sequentially immunoprecipitated with anti - HA agarose followed by anti - Flag agarose . PCR signals were normalized to input . error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences compared with WT * , P < 0 . 05 ; * * , P < 0 .

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D ChDIP of Flag-H2B-SUMO in the indicated strains expressing both Flag-H2B and HA-SUMO. Chromatin samples were sequentially immunoprecipitated with anti-HA agarose followed by anti-Flag agarose. PCR signals were normalized to the input DNA. The error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01


E ChDIP of Flag - H2B - SUMO in indicated mutant strains during CUP1 gene induction CUP1 induction was performed CUP1 OFF ON indicate uninduced induced ( 5 min ) conditions , respectively . error bars represent SD from three independent ChDIP assays . Data information : Asterisks indicate significant differences between uninduced induced in ( E ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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E ChDIP of Flag-H2B-SUMO in the indicated mutant strains during CUP1 gene induction The CUP1 induction was performed CUP1 OFF and ON indicate uninduced and induced (5 min) conditions, respectively. The error bars represent the SD from three independent ChDIP assays. Data information: Asterisks indicate significant differences between uninduced and induced cells in (E) (*, P <0.05; **, P <0.01).


F Immunoblot of Flag - tagged Ulp2 in indicated strains . Anti - PGK blotting shows similar loading .

F Immunoblot assay of Flag-tagged Ulp2 in the indicated strains. Anti-PGK blotting shows similar loading.


ChIP assays in indicated strains expressing Flag - tagged Ulp2 error bars indicate SD from three experiments . Data information : Asterisks indicate statistically significant differences compared with WT * , P < 0 . 05 ; * * , P < 0 .

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ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01


ChIP assays in indicated strains expressing Flag - tagged Ulp2 error bars indicate SD from three experiments . Data information : Asterisks indicate statistically significant differences compared with WT ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT (*, P <0.05; **, P <0.01).


A of CTD S2 - P relative to Rpb3 at 3 ' ORF regions was analyzed by ChIP using anti - S2 - P anti - Rpb3 antibodies . error bars represent SD from triplicate experiments . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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A The ratio of CTD S2-P relative to Rpb3 at 3' ORF regions was analyzed by ChIP using anti-S2-P and anti-Rpb3 antibodies. The error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


B A ulp2Δ single mutant three isolates of ulp2Δ ctk1Δ with YCplac33 - ULP2 were grown on SD - Ura or SD + 5 - FOA plates at 30 ° C for 2 - 3 days .

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B A ulp2Δ single mutant and three isolates of ulp2Δ ctk1Δ with YCplac33-ULP2 were grown on SD-Ura or SD+5-FOA plates at 30°C for 2-3 days.


E ChIP analyses of Ctk1 - TAP in indicated strains . ORF of TFIID - dominated genes indicates position 2 of PCR fragments from UTH1 ATP2 genes Error bars represent SD from triplicate experiments . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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E ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes Error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


F Co - IP of Ctk1 histone H2B interaction . WT ulp2Δ strains expressing indicated tagged were precipitated with IgG - Sepharose . IP INPUT were subjected to immunoblotting with PAP ( Ctk1 - TAP ) anti - Flag ( Flag - H2B ) antibodies . signals were quantified relative to PGK then normalized to WT value .

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F Co-IP analysis of Ctk1 and histone H2B interaction. WT and ulp2Δ strains expressing the indicated tagged proteins were precipitated with IgG-Sepharose. IP and INPUT proteins were subjected to immunoblotting with PAP (Ctk1-TAP) and anti-Flag (Flag-H2B) antibodies. Protein signals were quantified relative to PGK and then normalized to the WT value.


G ChDIP of Flag - H2B - SUMO in indicated strains " Pro " " ORF " of UTH1 ATP2 genes indicate positions 1 2 of PCR fragments , respectively error bars indicate SD from three independent ChDIPs . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

G ChDIP of Flag-H2B-SUMO in the indicated strains The "Pro" and "ORF" of UTH1 and ATP2 genes indicate positions 1 and 2 of PCR fragments, respectively The error bars indicate the SD from three independent ChDIPs. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


H Sensitivity to 6 - AU of indicated htb1 - 1 htb2 - 1 ( null alleles for both chromosomal H2B genes ) strains carrying pRS314 plasmids that express Flag - H2B or 2SUMO - H2B .

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H Sensitivity to 6-AU of indicated htb1-1 htb2-1 (null alleles for both chromosomal H2B genes) strains carrying pRS314 plasmids that express Flag-H2B or 2SUMO-H2B.


I , J ChIP analyses of indicated strains Occupancy of Rpb3 ( I ) of CTD S2 - P relative to Rpb3 ( J ) at 3 ' ORF regions of indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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I, J ChIP analyses of the indicated strains Occupancy of Rpb3 (I) and the ratio of CTD S2-P relative to Rpb3 (J) at 3' ORF regions of the indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


K ChIP analyses of Ctk1 - TAP in indicated strains . ORF of TFIID - dominated genes indicates position 2 of PCR fragments from UTH1 ATP2 genes Error bars represent SD from triplicate experiments . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

K ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes Error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


L Immunoblot of TAP - tagged Ctk1 in extracts prepared from strains Anti - PGK blotting shows similar loading .

L Immunoblot analysis of TAP-tagged Ctk1 proteins in extracts prepared from the strains Anti-PGK blotting shows similar loading.


M , N ChIP analyses of indicated strains Occupancy of Rpb3 ( M ) of CTD S2 - P relative to Rpb3 ( N ) at 3 ' ORF regions of indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

M, N ChIP analyses of the indicated strains Occupancy of Rpb3 (M) and the ratio of CTD S2-P relative to Rpb3 (N) at 3' ORF regions of the indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


( D ) showing expression change of representative NF - κB target genes from genes with significantly changed expression ( fold change > 2 ) . data represent log2 ( gene FPKM ) normalized for each row . - seq data is from 2 independent experiments .

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(D) Heatmap showing the expression change of representative NF-κB target genes from the genes with significantly changed expression (fold change >2). The data represent the log2 (gene FPKM) normalized for each row. RNA-seq data is from 2 independent experiments.


( E ) Intracellular cytokine of human TNF - α IL - 2 in sh - NC sh - p38IP Jurkat E6 . 1 stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for 6 hours ( average frequency of positive YFP + in sh - NC without stimulation = 1 ) .

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(E) Intracellular cytokine staining assay of human TNF-α and IL-2 in sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for 6 hours (average frequency of positive staining YFP+ cells in sh-NC cells without stimulation = 1).


( F ) ELISA of IL - 2 IL - 6 production in culture supernatants of human PBMCs transfected with si - NC or si - p38IP , then stimulated with 10 μg / ml anti - CD3 plus 10 μg / ml anti - CD28 ( αCD3 / CD28 ) for 24 hours .

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(F) ELISA analysis of IL-2 and IL-6 production in the culture supernatants of human PBMCs transfected with si-NC or si-p38IP, and then stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 (αCD3/CD28) for 24 hours.


( G H ) Real - time PCR of TNF - α , IL - 6 IL - 1β ( G ) of IFN - β , ISG54 ISG56 ( H ) mRNA production in RAW264 . 7 transfected with si - NC si - mp38IP - 1 then stimulated with 500 ng / ml LPS for indicated times ( average mRNA production in si - NC at 0 hour = 1 ) .

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(G and H) Real-time PCR analysis of TNF-α, IL-6 and IL-1β (G) and of IFN-β, ISG54 and ISG56 (H) mRNA production in RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times (average mRNA production in si-NC cells at 0 hour = 1).


( I ) ELISA of TNF - α , IL - 6 , IL - 1β IFN - β production in culture supernatants of human PBMCs transfected with si - NC si - p38IP then stimulated with 1μg / mL LPS for 24 hours .

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(I) ELISA analysis of TNF-α, IL-6, IL-1β and IFN-β production in the culture supernatants of human PBMCs transfected with si-NC and si-p38IP and then stimulated with 1μg/mL LPS for 24 hours.


( A ) Immunoblot with indicated antibodies of total lysates of sh - NC sh - p38IP Jurkat E6 . 1 stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for indicated times . Actin served as a loading control ( throughout ) .

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(A) Immunoblot analysis with the indicated antibodies of total cell lysates of sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times. Actin served as a loading control (throughout).


( B ) Statistical of p65 translocation of sh - NC sh - p38IP Jurkat E6 . 1 T stimulated with 50 ng / ml PMA plus 1µM ionomycin for indicated times . p65 translocation was indicated by intensities between p65 whole p65 from each . * * * P < 0 . 001 ( two - unpaired t - , mean s . e . m . ) .

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(B) Statistical analysis of p65 nuclear translocation of sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated with 50 ng/ml PMA plus 1µM ionomycin for the indicated times. p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).


( C ) Luciferase reporter of sh - NC sh - p38IP Jurkat E6 . 1 transfected with NF - κB luc plus an internal control Renilla luciferase reporter for 36 hours , followed by stimulation with ( + ) or without ( - ) 50ng / ml PMA plus 1 µM ionomycin for 8 hours . * * * P < 0 . 001 ; n . s . , not significant ( two - unpaired t - , mean s . e . m . ) .

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(C) Luciferase reporter assay of sh-NC and sh-p38IP Jurkat E6.1 cells transfected with NF-κB luc plus an internal control Renilla luciferase reporter for 36 hours, followed by stimulation with ( + ) or without ( - ) 50ng/ml PMA plus 1 µM ionomycin for 8 hours. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).


( D , E ) Immunoblot with indicated antibodies of total lysates of RAW264 . 7 transfected with si - NC si - mp38IP - 1 then stimulated with 500 ng / ml LPS for indicated times .

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(D,E) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times.


( A ) Fragmentation spectrum of p38IP peptide identified in TAK1 immunoprecipitated from Jurkat TAg by liquid chromatography - tandem mass spectrometry . m / z , mass / charge .

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(A) Fragmentation spectrum of the p38IP peptide identified in TAK1 complex immunoprecipitated from Jurkat TAg cells by liquid chromatography-tandem mass spectrometry. m/z, mass/charge ratio.


( B ) HEK293T were transfected with YFP - p38IP , lysates aliquots were immunoprecipitated with IgG , anti - TAB1 , anti - TAB2 anti - TAK1 respectively , then immunoblotted with indicated antibodies .

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(B) HEK293T cells were transfected with YFP-p38IP, and the cell lysates aliquots were immunoprecipitated with IgG, anti-TAB1, anti-TAB2 and anti-TAK1 respectively, and then immunoblotted with the indicated antibodies.


( C ) sh - NC or sh - p38IP Jurkat E6 . 1 T were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for indicated times lysed , followed byTAK1 immunoprecipitation . TAK1 beads were washed three times with lysis buffer two times with kinase buffer , then resuspended in kinase buffer containing 100 μM ATP 1μg recombinant GST - IKKβ ( 111 - 250aa ) as substrate to perform in vitro kinase , then immunoblot with p - IKKβ antibody .

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(C) sh-NC or sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times and lysed, followed byTAK1 immunoprecipitation. TAK1 beads were washed three times with lysis buffer and two times with kinase buffer, then resuspended in kinase buffer containing 100 μM ATP and 1μg recombinant GST-IKKβ(111-250aa) protein as substrate to perform in vitro kinase assay, and then immunoblot analysis with p-IKKβ antibody.


( D ) HEK293T were transfected with cMyc vector ( - ) or increasing amounts of cMyc - p38IP ( wedge ) , 24 hours post transfection , lysates were immunoprecipitated with IgG or anti - TAB2 then immunoblotted with anti - TAK1 anti - TAB2 . Expression of cMyc - p38IP was determined in immunoblotted lysates with anti - cMyc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) HEK293T cells were transfected with cMyc vector ( - ) or increasing amounts of cMyc-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAB2 and then immunoblotted with anti-TAK1 and anti-TAB2. Expression of cMyc-p38IP was determined in the immunoblotted lysates with anti-cMyc.


( E ) HEK293T were transfected with HA vector ( - ) or increasing amount of HA - p38IP ( wedge ) , 24 hours post transfection , lysates were immunoprecipitated with IgG or anti - TAK1 then immunoblotted with anti - TAB2 , anti - TAB1 , anti - HA anti - TAK1 . Expression of HA - p38IP was determined in immunoblotted lysates with anti - HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) HEK293T cells were transfected with HA vector ( - ) or increasing amount of HA-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAK1 and then immunoblotted with anti-TAB2, anti-TAB1, anti-HA and anti-TAK1. Expression of HA-p38IP was determined in the immunoblotted lysates with anti-HA.


( F ) sh - NC sh - p38IP Jurkat E6 . 1 T were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for indicated times , lysates were immunoprecipitated with anti - TAB2 immunoblotted with anti - TAK1 anti - TAB2 .

(F) sh-NC and sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and the cell lysates were immunoprecipitated with anti-TAB2 and immunoblotted with anti-TAK1 and anti-TAB2.


( H ) Jurkat TAg T transfected with cMyc vector or cMyc - p38IP were stimulated with 10μg / ml anti - CD3 for indicated times , then were lysed immunoprecipitated with anti - TAK1 antibody immunoblotted with anti - K63Ub , anti - cMyc anti - TAK1 antibodies . Lysates were blotted with indicated antibodies , actin served as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Jurkat TAg T cells transfected with cMyc vector or cMyc-p38IP were stimulated with 10μg/ml anti-CD3 for the indicated times, and then were lysed and immunoprecipitated with anti-TAK1 antibody and immunoblotted with anti-K63Ub, anti-cMyc and anti-TAK1 antibodies. Lysates were blotted with the indicated antibodies, and actin served as the loading control.


( I ) sh - NC sh - p38IP Jurkat E6 . 1 T stimulated as in ( F ) were lysed immunoprecipitated with anti - TAK1 immunoblotted with anti - K63Ub anti - TAK1 . Lysates were immunoblotted with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-K63Ub and anti-TAK1. Lysates were immunoblotted with the indicated antibodies.


( J ) Jurkat TAg T transfected stimulated as in ( H ) were lysed immunoprecipitated with anti - TAB2 immunoblotted with anti - K63Ub anti - TAB2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Jurkat TAg T cells transfected and stimulated as in (H) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2.


( K ) sh - NC sh - p38IP Jurkat E6 . 1 stimulated as in ( F ) were lysed immunoprecipitated with anti - TAB2 immunoblotted with anti - K63Ub anti - TAB2 . Data ( B - F , H - K ) are representatives of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) sh-NC and sh-p38IP Jurkat E6.1 cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2. Data (B-F, H-K) are representatives of at least three independent experiments.


( A ) Jurkat E6 . 1 T were stimulated with 10 μg / ml anti - CD3 2 μg / ml anti - CD28 for indicated times , lysates were immunoprecipitated immunoblotted as indicated . relative of IP bands was quantified by densitometry is shown below graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for the indicated times, the cell lysates were immunoprecipitated and immunoblotted as indicated. The relative intensity of the IP bands was quantified by densitometry and is shown below the graph.


( B ) HEK293T transfected with Flag - tagged TAB2 ( open arrow ) or TAB2 - ub ( solid arrow ) were lysed immunoprecipitated with anti - Flag , then immunoblotted with indicated antibodies . asterisk indicates a non - specific band . relative of IP bands was quantified by densitometry is presented in right .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) HEK293T cells transfected with Flag-tagged TAB2 (open arrow) or TAB2-ub (solid arrow) were lysed and immunoprecipitated with anti-Flag, and then immunoblotted with the indicated antibodies. The asterisk indicates a non-specific band. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.


( C ) HEK293T co - transfected with HA - tagged p38IP together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) were lysed immunoprecipitated with anti - cMyc , then immunoblotted with indicated antibodies . Asterisk indicates non - specific bands . relative of IP bands was quantified by densitometry is presented in right .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HEK293T cells co-transfected with HA-tagged p38IP together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow) were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. Asterisk indicates non-specific bands. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.


( D ) HEK293T co - transfected with HA - tagged p38IP , Flag - tagged TAB2 - ub , together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) , were lysed immunoprecipitated with anti - cMyc , then immunoblotted with indicated antibodies . relative of IP bands was quantified by densitometry is presented in right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) HEK293T cells co-transfected with HA-tagged p38IP, Flag-tagged TAB2-ub, together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow), were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.


( A ) GSH - Sepharose beads - immobilized full or truncated GST - TAK1 were incubated with K63 - linked polyUb chains in 300 μl binding buffer , followed by washing resolving in SDS - PAGE , then immunoblotted with anti - ub . GST - fused NEMO serve as positive control NEMO L329P mutant as negative control . blotted was stained with Ponceau S to demonstrate amount of input GST - fused ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GSH-Sepharose beads-immobilized full length or truncated GST-TAK1 proteins were incubated with K63-linked polyUb chains in 300 μl binding buffer, followed by washing and resolving in SDS-PAGE, and then immunoblotted with anti-ub. GST-fused NEMO serve as positive control and NEMO L329P mutant as negative control. The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST-fused proteins (lower panel).


( C D ) Immobilized GST - TAB2 was pre - incubated with K63 - linked polyUb chains then eluted from beads , followed by incubation with eluted GST - TAK1 GST - p38IP as shown in ( B ) . Incubation mixture were immunoprecipitated with anti - TAB2 or anti - TAK1 , then immunoblotted with anti - ub , anti - TAB2 , anti - TAK1 anti - p38IP . ( E ) Statistical of polyUb binding to TAB2 or TAK1 in ( C D ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - unpaired t - , mean s . e . m . ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C and D) Immobilized GST-TAB2 was pre-incubated with K63-linked polyUb chains and then eluted from beads, followed by incubation with eluted GST-TAK1 and GST-p38IP as shown in (B). Incubation mixture were immunoprecipitated with anti-TAB2 or anti-TAK1, and then immunoblotted with anti-ub, anti-TAB2, anti-TAK1 and anti-p38IP. (E) Statistical analysis of polyUb binding to TAB2 or TAK1 in (C and D). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).


( F ) Immobilized GST - TAB2 GST - TAK1 were respectively pre - incubated with K63 - linked polyUb chains or not as indicated , then eluted from beads , followed by coincubation as indicated . Incubation mixture were immunoprecipitated with anti - TAB2 , then immunoblotted with anti - TAB2 anti - TAK1 . relative of immunoprecipitated TAK1 was calculated shown in right . * * * P < 0 . 001 ; n . s . , not significant ( two - unpaired t - , mean s . e . m . ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immobilized GST-TAB2 and GST-TAK1 were respectively pre-incubated with K63-linked polyUb chains or not as indicated, then eluted from beads, followed by coincubation as indicated. Incubation mixture were immunoprecipitated with anti-TAB2, and then immunoblotted with anti-TAB2 and anti-TAK1. The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).


( G ) Immobilized GST - TAK1 was pre - incubated with K63 - linked polyUb chains or not , then eluted from beads , followed by incubation with eluted GST - p38IP . Incubation mixture were immunoprecipitated with anti - TAK1 , then immunoblotted with anti - TAK1 anti - p38IP . relative of immunoprecipitated p38IP was calculated shown in right . * * * P < 0 . 001 ( two - unpaired t - , mean s . e . m . ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immobilized GST-TAK1 was pre-incubated with K63-linked polyUb chains or not, then eluted from beads, followed by incubation with eluted GST-p38IP. Incubation mixture were immunoprecipitated with anti-TAK1, and then immunoblotted with anti-TAK1 and anti-p38IP. The relative intensity of immunoprecipitated p38IP was calculated and shown in the right. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).


( B ) HEK293T transfected with N - terminal ( left panels ) or C - terminal ( right panels ) deletion mutants of p38IP as indicated in ( A ) were lysed after 24 hours transfection immunoprecipitated with IgG or anti - TAK1 , followed by incubation with G beads , then immunoblotted with anti - YFP anti - TAK1 .

(B) HEK293T cells transfected with N-terminal (left panels) or C-terminal (right panels) deletion mutants of p38IP as indicated in (A) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.


( D ) GST pull down . 3μg recombinant GST - T1B which immobilized on glutathione Sepharose beads was incubated with lysates that obtained by adding 1ml lysis buffer to HEK293T in 10 cm culture dish with a final around 1μg / μl , after 4 hrs incubation , washed beads with lysis buffer five times subjected to SDS - PAGE for immunoblotting with anti - TAK1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) GST pull down assay. 3μg recombinant GST-T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg/μl, after 4 hrs incubation, washed beads with cell lysis buffer five times and subjected to SDS-PAGE for immunoblotting with anti-TAK1.


( E ) HEK293T transfected with YFP - p38IP T1B were lysed after 24 hours transfection immunoprecipitated with IgG or anti - TAK1 , followed by incubation with G beads , then immunoblotted with anti - YFP anti - TAK1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) HEK293T cells transfected with YFP-p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.


( F ) Jurkat TAg transfected with YFP vector or YFP - T1B were stimulated with 10μg / ml anti - CD3 for indicated times , then lysed immunoprecipitated with anti - TAK1 , followed by immunoblotting with indicated antibodies .

(F) Jurkat TAg cells transfected with YFP vector or YFP-T1B were stimulated with 10μg/ml anti-CD3 for indicated times, then lysed and immunoprecipitated with anti-TAK1, followed by immunoblotting with the indicated antibodies.


( G ) human PBMCs transfected with YFP vector or YFP - T1Bwere stimulated with 10 μg / ml anti - CD3 plus 10 μg / ml anti - CD28 for indicated times , then lysed analyzed by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Primary human PBMCs transfected with YFP vector or YFP-T1Bwere stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 for the indicated times, then lysed and analyzed by immunoblotting.


( H ) Intracellular cytokine of TNF - α ( upper ) IL - 2 ( lower ) in human PBMCs transfected stimulated as in ( F ) for 3 hours ( TNF - α ) 8 hours ( IL - 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Intracellular cytokine staining analysis of TNF-α (upper) and IL-2 (lower) in primary human PBMCs transfected and stimulated as in (F) for 3 hours (TNF-α) and 8 hours (IL-2).


( A ) HEK293T co - transfected with YFP - p38IP Flag - vector , Flag - USP3 or Flag - USP4 as indicated were lysed immunoprecipitated with anti - Flag antibody immunoblotted with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HEK293T cells co-transfected with YFP-p38IP and Flag-vector, Flag-USP3 or Flag-USP4 as indicated were lysed and immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.


( B ) HEK293T transfected with Flag - USP4 YFP - vector or YFP - p38IP as indicated were lysed immunoprecipitated with anti - IgG or anti - TAK1 then immunoblotted with anti - Flag anti - TAK1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) HEK293T cells transfected with Flag-USP4 and YFP-vector or YFP-p38IP as indicated were lysed and immunoprecipitated with anti-IgG or anti-TAK1 and then immunoblotted with anti-Flag and anti-TAK1.


( C ) HEK293T transfected with plasmids as indicated were lysed immunoprecipitated with anti - TAK1 , followed by in vivo ubiquitination of TAK1 as described in methods .

(C) HEK293T cells transfected with plasmids as indicated were lysed and immunoprecipitated with anti-TAK1, followed by in vivo ubiquitination assay of TAK1 as described in methods.


( D ) In vitro deubiquitination of TAK1 as described in methods .

(D) In vitro deubiquitination assay of TAK1 as described in methods.


( E ) sh - NC sh - p38IP Jurkat E6 . 1 transfected with si - NC , si - p38IP , Flag or Flag - USP4 as indicated were stimulated with anti - CD3 / CD28 for 0 15 min at 48 hours post transfection , then lysed immunoprecipitated with anti - TAK1 under stringent conditions immunoblotted with anti - ub . Statistical of p - p38 was shown below blot . * * P < 0 . 01 ; n . s . , not significant ( two - unpaired t - , mean s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) sh-NC and sh-p38IP Jurkat E6.1 cells transfected with si-NC, si-p38IP, Flag or Flag-USP4 as indicated were stimulated with anti-CD3/CD28 for 0 and 15 min at 48 hours post transfection, and then lysed and immunoprecipitated with anti-TAK1 under stringent conditions and immunoblotted with anti-ub. Statistical analysis of p-p38 levels was shown below the blot. **P < 0.01; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).


( F ) Jurkat E6 . 1 T were stimulated with anti - CD3 / CD28 for indicated times . lysates were immunoprecipitated with anti - p38IP or anti - TAK1 then immunoblotted with indicated antibodies .

(F) Jurkat E6.1 T cells were stimulated with anti-CD3/CD28 for the indicated times. The cell lysates were immunoprecipitated with anti-p38IP or anti-TAK1 and then immunoblotted with the indicated antibodies.


( G ) sh - NC sh - p38IP Jurkat E6 . 1 were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for indicated times , then lysed immunoprecipitated with anti - TAK1 immunoblotted with anti - USP4 anti - TAK1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) sh-NC and sh-p38IP Jurkat E6.1 cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and then lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-USP4 and anti-TAK1.


( H ) Mapping USP4 binding domain of p38IP .

(H) Mapping the USP4 binding domain of p38IP.


, 12wk wildtype ( WT ; C57Bl / 6J ) or obese / diabetic monogenic ( db / db ; BKS . Cg - m + / + Lepr DB / J ; N = 4 / group , A - E ) , New Zealand Black ( NZB ) polygenic obese / prediabetic New Zealand Obese ( NZO ; n = 4 / group ; F - J ) , as well as young ( i . e . 3 mo ) aged ( i . e . 22 mo ; n = 5 / group ; K - O ) , mice were fed ad libitum ( fed ) or fasted for 24h ( fasted ) . Serum non - esterified fatty ( A , F , K ) , triglycerides ( B , G , L ) ketone bodies ( C , H , M ) were measured . In addition , serumacylcarinitine profiling was conducted medium - chain ( D , I , N ) long - chain ( E , J , O ) acylcarnitine concentrations are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O

Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic monogenic (db/db; BKS.Cg-m+/+ Lepr DB/J; N = 4/group, A-E), New Zealand Black (NZB) and polygenic obese/prediabetic New Zealand Obese (NZO; n=4/group; F-J), as well as young (i.e. 3 mo) and aged (i.e.22 mo; n = 5/group; K-O), mice were fed ad libitum (fed) or fasted for 24h (fasted). Serum non-esterified fatty acids (A, F, K), triglycerides (B, G, L) and ketone bodies (C, H, M) were measured. In addition, serumacylcarinitine profiling was conducted and medium-chain (D, I, N) and long-chain (E, J, O) acylcarnitine concentrations are shown.


In another cohort of mice , ex vivo long - chain fatty acid ( LCFA ) metabolism , including uptake ( P ) , oxidation ( Q ) non - oxidative LCFA disposal ( NOFAD ; R ) , in precision cut liver slices from fed fasted WT db / db mice ( n = 3 / group ; 4 slices per mouse ) , were determined . In addition , in slices from fasted mice , glucose output was determined in presence of incubation with NEFA ( BSA - NEFA ) or vehicle ( BSA ) ( S ) .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O

In another cohort of mice, ex vivo long-chain fatty acid (LCFA) metabolism, including uptake (P), oxidation (Q) and non-oxidative LCFA disposal (NOFAD; R), in precision cut liver slices from fed and fasted WT and db/db mice (n = 3/ group; 4 slices per mouse), were determined. In addition, in slices from fasted mice, glucose output was determined in the presence of incubation with NEFA (BSA-NEFA) or vehicle (BSA) (S).


Liver mRNA expression of Growth Arrest Damage inducible 45 alpha ( Gadd45a ; A , D , G ) , beta ( Gadd45b ; B , E , H ) gamma ( Gadd45g ; C , F , I ) was measured in fed fasted obese / diabetic monogenic ( db / db ( n = 4 / group ) ; A , - C ) , obese / pre - diabetic polygenic New Zealand Obese ( NZO ( n = 4 / group ; D - F ) , aged C57Bl / 6J ( 22mo ; n = 5 / group ; G - I ) as well as corresponding lean , young wildtype ( WT ) mice ( n = 4 / group ) . Matched controls for NZO aged mice were New Zealand Black ( NZB ) 12wk old C57Bl / 6J ( 12wk ) mice , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Liver mRNA expression of Growth Arrest and DNA Damage inducible 45 alpha (Gadd45a; A, D, G), beta (Gadd45b; B, E, H) and gamma (Gadd45g; C, F, I) was measured in fed and fasted obese/diabetic monogenic (db/db (n=4/group); A,-C), obese/pre-diabetic polygenic New Zealand Obese (NZO (n=4/group; D-F), and aged C57Bl/6J (22mo; n = 5/group; G-I) as well as corresponding lean, young wildtype (WT) mice (n=4/group). Matched controls for the NZO and aged mice were New Zealand Black (NZB) and 12wk old C57Bl/6J (12wk) mice, respectively.


Liver GADD45β as well as housekeeping Valosin containing ( VCP ) expression was measured from liver samples ( J - L ) . Data are mean ± . Effect of genotype , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Effect of nutritional state : # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 . statistical used respective p - value outputs can be found in Appendix Table S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Liver GADD45β as well as the housekeeping protein Valosin containing protein (VCP) protein expression was measured from liver samples (J-L). Data are mean ± SEM. Effect of genotype, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001. The statistical test used and respective p-value outputs can be found in Appendix Table S1.


, GADD45β + / + ( WT ; n = 6 ) or GADD45β - / - mice ( KO ; n = 4 ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) , subsequently refed for 24h . O2 consumption rate ( A ) , CO2 production rate ( B ) respiratory exchange ( C ) were measured by indirect calorimetry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

Male, GADD45β+/+ (WT; n=6) or GADD45β-/- mice (KO; n=4) were fed ad libitum (fed) or fasted for 24h (fasted), and subsequently refed for 24h. O2 consumption rate (A), CO2 production rate (B) and respiratory exchange ratio (C) were measured by indirect calorimetry.


In a distinct cohort , GADD45β + / + ( WT ; n = 5 ) or GADD45β - / - mice ( KO ; n = 8 ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) . Serumnon - esterified fatty ( D ) ketone bodies ( E ) as well as serum ( F ) liver ( G ) triglycerides ( TG ) were measured .

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In a distinct cohort, male GADD45β+/+ (WT; n=5) or GADD45β-/- mice (KO; n= 8) were fed ad libitum (fed) or fasted for 24h (fasted). Serumnon-esterified fatty acids (D) and ketone bodies (E) as well as serum (F) and liver (G) triglycerides (TG) were measured.


Serum TG ( H ) , NEFA ( I ) , blood glucose ( BG , J ) concentrations during an oral lipid tolerance in overnight fasted GADD45β + / + ( WT ; n = 5 ) or GADD45β - / - ( KO ; n = 5 ) mice .

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Serum TG (H), NEFA (I), and blood glucose (BG, J) concentrations during an oral lipid tolerance test in overnight fasted GADD45β+/+ (WT; n = 5) or GADD45β-/- (KO; n=5) mice.


Bloodglucose excursion during intraperitoneal insulin tolerance ( K ) as well as fasting bloodglucose ( L ) , seruminsulin ( M ) , HOMA - IR ( N ) in GADD45β + / + ( WT ; n = 6 ) or GADD45β - / - mice ( KO , n = 9 ) chronically fed a normal - ( NFD ) or high ( HFD ) fat diet . Data are mean ± .

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Bloodglucose excursion during and intraperitoneal insulin tolerance test (K) as well as fasting bloodglucose (L), seruminsulin (M), and HOMA-IR (N) in GADD45β+/+ (WT; n = 6) or GADD45β-/- mice (KO, n = 9) chronically fed a normal- (NFD) or high (HFD) fat diet. Data are mean ± SEM.


, GADD45β + / + ( WT ) or GADD45β - / - mice ( KO ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) ex vivo liver slice long - chain fatty acid ( LCFA ) metabolism was measured including LCFA uptake ( A ) , oxidation ( B ) non - oxidative LCFA disposal ( NOFAD ) was calculated ( C ) .

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Male, GADD45β+/+ (WT) or GADD45β-/- mice (KO) were fed ad libitum (fed) or fasted for 24h (fasted) and ex vivo liver slice long-chain fatty acid (LCFA) metabolism was measured including LCFA uptake (A), oxidation (B) and non-oxidative LCFA disposal (NOFAD) was calculated (C).


In addition , glucose production was measured ( D ) in presence ( BSA - NEFA ) or absence ( BSA ) of extracellular fatty ( n = 4 / group with 4 liver slices per mouse liver ) .

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In addition, glucose production was measured (D) in the presence (BSA-NEFA) or absence (BSA) of extracellular fatty acids (n=4/group with 4 liver slices per mouse liver).


Gadd45b mRNA expression was measured from fractionated parenchymal hepatocytes ( Hepas ) as well as non - parenchymal ( NP ) from C57Bl / 6J mice fed ad libitum ( fed ) or fasted for 24h ( fasted ) ; n = 3 / group ( E ) .

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Gadd45b mRNA expression was measured from fractionated parenchymal hepatocytes (Hepas) as well as non-parenchymal cells (NP) from C57Bl/6J mice fed ad libitum (fed) or fasted for 24h (fasted); n=3/group (E).


, C57Bl / 6J mice with ( AAV - G45b miR ) or without ( AAV - NC miR ) liver - hepatocyte - restricted GADD45β silencing were fed or fasted for 24h ( n = 6 / group ) serum of non - esterified fatty ( NEFA ; F ) as well as liver triglyceride ( TG ) ( G ) were measured .

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Male, C57Bl/6J mice with (AAV-G45b miR) or without (AAV-NC miR) liver-hepatocyte-restricted GADD45β silencing were fed or fasted for 24h (n = 6/group) and serum levels of non-esterified fatty acids (NEFA; F) as well as liver triglyceride (TG) concentration (G) were measured.


, GADD45β + / + ( WT ; n = 16 ) or GADD45β - / - mice ( KO ; n = 15 ) fasted for 24h ( fasted ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 7 - 8 / group ) . LiverTG ( H ) was measured .

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Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). LiverTG concentration (H) was measured.


, C57Bl / 6J mice with ( AAV - G45b miR ; n = 15 ) or without ( AAV - NC miR , n = 13 ) liver - hepatocyte - restricted GADD45β silencing were chronically fed a normal ( NFD ) or high ( HFD ) fat diet ( n = 6 - 8 / group ) . Fasting blood glucose ( I ) serum insulin ( J ) were measured from which HOMA - IR was calculated ( K ) .

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Male, C57Bl/6J mice with (AAV-G45b miR; n= 15) or without (AAV-NC miR, n = 13) liver-hepatocyte-restricted GADD45β silencing were chronically fed a normal (NFD) or high (HFD) fat diet (n = 6-8/group). Fasting blood glucose (I) and serum insulin (J) were measured from which HOMA-IR was calculated (K).


, 12wk wildtype ( WT ; C57Bl / 6J ) or obese / diabetic ( db / db ; BKS . Cg - m + / + Lepr DB / J ) with ( AD - G45b OE ) or without ( AD - NC ) prior liver - restricted GADD45β over - expression were fasted blood glucose ( A ) serum insulin ( B ) were measured from which HOMA - IR was calculated ( C ) ( n = 4 - 6 / group ) . Data are mean ± . Effect of genotype / viral manipulation , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Effect of nutritional state : # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 .

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Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) prior liver-restricted GADD45β over-expression were fasted and blood glucose ( A) and serum insulin (B) were measured from which HOMA-IR was calculated (C) (n = 4-6/group). Data are mean ± SEM. Effect of genotype/viral manipulation, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001.


Liver GADD45B mRNA expression from men with ( T2D ) or without ( NGT ) type 2 diabetes ( n = 14 - 23 / group ) .

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Liver GADD45B mRNA expression from men with (T2D) or without (NGT) type 2 diabetes (n = 14-23/group).


Scatter plots fasting plasma triglycerides ( E ) glucose ( F ) in correlation with liver GADD45B mRNA expression ( n = 37 ) . Inserts show r2 values p values from Spearman ' s correlation . statistical used respective p - value outputs can be found in Appendix Table S1 .

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Scatter plots fasting plasma triglycerides (E) and glucose (F) in correlation with liver GADD45B mRNA expression (n = 37). Inserts show r2 values and p values from Spearman's correlation test. The statistical test used and respective p-value outputs can be found in Appendix Table S1.


, GADD45β + / + ( WT ; n = 16 ) or GADD45β - / - mice ( KO ; n = 15 ) fasted for 24h ( fasted ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 7 - 8 / group ) . Liver mRNA expression of Gadd45b ( A ) as well as fatty acid metabolic genes ( B ) encompassing transport ( Slc28a2 , Slc27a5 , Cd36 ) , intracellular binding ( Fabp1 , Dbi ) metabolism ( Acly , Dgat1 , Atgl , Hsl ) .

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Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). Liver mRNA expression of Gadd45b (A) as well as fatty acid metabolic genes (B) encompassing transport (Slc28a2, Slc27a5, Cd36), intracellular binding (Fabp1, Dbi) and metabolism (Acly, Dgat1, Atgl, Hsl).


C : Representative immunoblots of FATP2 , CD36 , FABP1 GADD45β from liver whole tissue lysate ( W ) as well as fractionated organelles / intraceuular structures including nuclei ( N ) , mitochondria ( MT ) , microsomes ( MS ) cytoplasm ( C ) , from GADD45β + / + ( WT ) GADD45β - / - ( KO ) mice . D : Quantified band densities of FABP1 enrichment from fractions in C ( n = 4 / group ) .

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C: Representative immunoblots of FATP2, CD36, FABP1 and GADD45β from liver whole tissue lysate (W) as well as fractionated organelles/intraceuular structures including nuclei (N), mitochondria (MT), microsomes (MS) and cytoplasm (C), from GADD45β+/+ (WT) and GADD45β-/- (KO) mice. D: Quantified band densities of FABP1 enrichment from fractions in C (n = 4/group).


E : Liver fraction enrichment of FABP1 from , GADD45β + / + ( WT ) or GADD45β - / - ( KO ) fasted for 24h with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 4 / group ) . Insert shows a representative FABP1 immunoblot .

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E: Liver fraction enrichment of FABP1 from male, GADD45β+/+ (WT) or GADD45β-/- (KO) fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot.


F : Liver fraction enrichment of FABP1 from obese / diabetic db / db mice fasted for 24h with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 4 / group ) . Insert shows a representative FABP1 immunoblot .

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F: Liver fraction enrichment of FABP1 from obese/diabetic male db/db mice fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot.


G : FABP1 GADD45B immunoblots from Flag - immunoprecipitations ( IP - FLAG ) or mock IP ( IP - HA ) from liver input samples from mice with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression . Shown is a representative immunoblot from 3 separate experiments using 3 different input samples per condition .

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G: FABP1 and GADD45B immunoblots from Flag-immunoprecipitations (IP-FLAG) or mock IP (IP-HA) from liver input samples from mice with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression. Shown is a representative immunoblot from 3 separate experiments using 3 different input samples per condition.


H : Liver long chain acyl - CoA ( LC - acyl - CoA ) concentrations were determined in GADD45β + / + ( WT ) or GADD45β - / - ( KO ) mice ( H ; n = 6 / group ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( I ; n = 5 / group ) .

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H: Liver long chain acyl-CoA (LC-acyl-CoA) concentrations were determined in GADD45β+/+ (WT) or GADD45β-/- (KO) mice (H; n=6/ group) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (I; n =5/group).


Liver LC - acyl - CoA concentrations were determined in wildtype ( WT ; C57Bl / 6J ) or obese / diabetic ( db / db ; BKS . Cg - m + / + Lepr DB / J ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( J ; n = 4 / group ) .

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Liver LC-acyl-CoA concentrations were determined in wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (J; n=4/group).


B Identification of sec homozygotes . LB represents left border primer of T - insertion . LP RP represent left right genomic primers , respectively .

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B Identification of sec homozygotes. LB represents the left border primer of the T-DNA insertion. LP and RP represent the left and right genomic primers, respectively.


C qRT - PCR of SEC mRNA in sec - 4 sec - 5 mutants . expression level was normalized to that of TUBULIN , a reference gene for qRT - PCR . Independent biological experiments were repeated three times , one representative result is shown here . Data shown are means ± standard deviation ( s . d . ) , n = 3 .

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C qRT-PCR analysis of SEC mRNA levels in sec-4 and sec-5 mutants. The expression level was normalized to that of TUBULIN, a reference gene for qRT-PCR. Independent biological experiments were repeated three times, and one representative result is shown here. Data shown are means ± standard deviation (s.d.), n = 3.


D early flowering phenotype of sec - 5 mutant under long - day ( LD ) conditions . Scale bar : 1 cm .

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D The early flowering phenotype of sec-5 mutant under long-day (LD) conditions. Scale bar: 1 cm.


E qRT - PCR of expression of FLC , SOC1 , MAF1 - 3 in 12 - d - old vegetative Col - 0 , sec - 4 sec - 5 . Independent biological experiments were repeated three times , one representative result is shown here . expression level was normalized to that of TUBULIN . Data are means ± s . d . , n = 3 . ( * ) , Statistical significance ( two - t - ) with P < 0 . 05 .

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E qRT-PCR analysis of the expression levels of FLC, SOC1, and MAF1-3 in 12-d-old vegetative Col-0, sec-4 and sec-5 plants. Independent biological experiments were repeated three times, and one representative result is shown here. The expression level was normalized to that of TUBULIN. Data are means ± s.d., n = 3. (*), Statistical significance (two-tailed t-test) with P < 0.05.


F sec - 5 mutant exhibits an early flowering phenotype under short - day ( SD ) conditions . Scale bar : 1 cm .

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F The sec-5 mutant exhibits an early flowering phenotype under short-day (SD) conditions. Scale bar: 1 cm.


G qRT - PCR of FLC SOC1 expression in Col - 0 sec - 5 under SD conditions . expression level was normalized to that of TUBULIN . Experiments were repeated three times , one representative result is shown here . Data are mean ± s . d . , n = 3 . ( * ) , Statistical significance ( two - t - ) with P < 0 . 05 .

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G qRT-PCR analysis of FLC and SOC1 expression in Col-0 and sec-5 plants under SD conditions. The expression level was normalized to that of TUBULIN. Experiments were repeated three times, and one representative result is shown here. Data are mean ± s.d., n = 3. (*), Statistical significance (two-tailed t-test) with P < 0.05.


B ChIP - qPCR of H3K4me2 of indicated regions at FLC . C ChIP - qPCR of H3K4me3 of indicated regions at FLC . D ChIP - qPCR of H3K36me3 of indicated regions at FLC . E ChIP - qPCR of H3K27me3 of indicated regions at FLC . Data information : For ChIP , 12 - d - old were collected three independent experiments were conducted . Each bar represents mean ± s . d . of three independent experiments , n = 3 . relative abundance was normalized to input .

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B ChIP-qPCR assay of H3K4me2 levels of indicated regions at FLC chromatin. C ChIP-qPCR assay of H3K4me3 levels of indicated regions at FLC chromatin. D ChIP-qPCR assay of H3K36me3 levels of indicated regions at FLC chromatin. E ChIP-qPCR assay of H3K27me3 levels of indicated regions at FLC chromatin .Data information: For ChIP analysis, 12-d-old plants were collected and three independent experiments were conducted. Each bar represents the mean ± s.d. of three independent experiments, n=3. The relative abundance was normalized to the input.


A Yeast two - hybrid of interaction between ATX1 SEC . full - coding ( CDS ) of ATX1 was cloned into bait vector pGBKT7 . A full - SEC CDS a truncated fragment were cloned into prey vector pGADT7 to express complete or truncated SEC - N ( including only TPR domains at N - terminus ) , respectively . AD vectors expressing SEC or SEC - N were each cotransformed with BD - ATX1 . SD / 2 , SD / - Leu / - Trp medium ; SD / 4 , SD / - Ade / - His / - Leu / - Trp medium . Cotransformed pGBKT7 - 53 / pGADT7 - T was used as a positive control .

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A Yeast two-hybrid assay of the interaction between ATX1 and SEC. The full-length coding sequence (CDS) of ATX1 was cloned into the bait vector pGBKT7. A full-length SEC CDS and a truncated fragment were cloned into the prey vector pGADT7 to express complete or truncated SEC-N (including only TPR domains at the N-terminus), respectively. AD vectors expressing SEC or SEC-N were each cotransformed with BD-ATX1. SD/2, SD/-Leu/-Trp medium; SD/4, SD/-Ade/-His/-Leu/-Trp medium. Cotransformed pGBKT7-53/pGADT7-T was used as a positive control.


B Immunoblot for O - GlcNAcylation of ATX1 by SEC using anti - CTD110 . 6 antibody . HA - tagged ATX1 was expressed alone or together with Flag - tagged SEC in tobacco leaves . were extracted from tobacco mesophyll , expression of FLAG - SEC ATX1 - HA was confirmed by western blotting .

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B Immunoblot analysis for O-GlcNAcylation of ATX1 by SEC using anti-CTD110.6 antibody. HA-tagged ATX1 was expressed alone or together with Flag-tagged SEC in tobacco leaves. Nuclear proteins were extracted from tobacco mesophyll cells, and expression of FLAG-SEC and ATX1-HA was confirmed by western blotting.


C Flowering phenotype of 35S : : ATX1 - HA overexpression transgenic lines in Col - 0 sec - 5 backgrounds , respectively . Scale bar = 1 cm .

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C Flowering phenotype of 35S::ATX1-HA overexpression transgenic lines in Col-0 and sec-5 backgrounds, respectively. Scale bar = 1 cm.


D Immunoblot using anti - HA antibody to confirm ATX1 - HA expression in Col - 0 35S : : ATX1 - HA - overexpressing lines ACTIN was used as a loading control .

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D Immunoblot analysis using anti-HA antibody to confirm ATX1-HA expression in Col-0 and 35S::ATX1-HA-overexpressing lines ACTIN was used as a loading control.