Calcineurin activity Ca2 + responses simultaneously monitored in single RAW 247 . 6 using CaNAR2 jRGECO1a , respectively , upon addition of 3 - μm beads . CaNAR2 signals ( FRET Y / C ) were inhibited by FK506 ( 10 μM ) Ned - 19 ( 10 μM ) ; ( H ) single - traces ( I ) mean maximum responses . n = 65 - 169 ; * * * P < 0 . 001 vs control .

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Calcineurin activity and cytosolic Ca2+ responses simultaneously monitored in single RAW 247.6 using CaNAR2 and jRGECO1a, respectively, upon addition of 3-μm beads. CaNAR2 signals (FRET Y/C) were inhibited by FK506 (10 μM) and Ned-19 (10 μM); (H) single-cell traces and (I) mean maximum responses. n = 65-169; ***P<0.001 vs control.


Phagocytosis induced dephosphorylation of dynamin - 2 ( of phospho - dynamin to total dynamin - 2 expressed as a of WT at time zero ) in WT BMDM , but not in Tpcn1 - / - BMDM ; * * P < 0 . 01 , * * * P < 0 . 001 ( time - point matched unpaired t - ) n = 10 - 15 . Measured using in - western , representative wells of plate image : total dyn2 ( red ) phospho - dyn2 ( green ) . See also Appendix Figures S6 S7 .

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Phagocytosis induced dephosphorylation of dynamin-2 (ratio of phospho-dynamin to total dynamin-2 expressed as a percentage of WT at time zero) in WT BMDM, but not in Tpcn1-/- BMDM; **P<0.01, ***P<0.001 (time-point matched unpaired t-test) n = 10-15. Measured using in-cell western assay, representative wells of plate image: total dyn2 (red) and phospho-dyn2 (green). See also Appendix Figures S6 and S7.


RAW 246 . 7 macrophages expressing dynein - binding ( mTagBFP2 - BicD2 - FKBP12 ) for rapalog - induced lysosome repositioning to MTOC , also co - expressed HsLAMP1 - mCherry with or without a C - terminal FRB * ( LAMP - FRB * or LAMP , in red ) . were treated with 0 . 1 % ethanol or 250 nM rapalog for 2 h . Only tripartite of FKBP12 - rapalog - FRB induced clustering of lysosomes at MTOC ( A , B ) . This clustering was selective for lysosomes because ER mitochondria ( KDEL - GFP MitoTracker Green ) were not affected ( C ) . ( A , B ) Phagocytosis of IgG - 3 - μm beads was reduced by immobilizing lysosomes at MTOC , n = 80 - 564 ; * * * P < 0 . 001 .

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RAW 246.7 macrophages expressing the dynein-binding protein (mTagBFP2-BicD2-FKBP12) for rapalog-induced lysosome repositioning to the MTOC, also co-expressed HsLAMP1-mCherry with or without a C-terminal FRB* (LAMP-FRB* or LAMP, in red). Cells were treated with 0.1% ethanol or 250 nM rapalog for 2 h. Only the tripartite complex of FKBP12-rapalog-FRB induced clustering of lysosomes at the MTOC (A, B). This clustering was selective for lysosomes because ER and mitochondria (KDEL-GFP and MitoTracker Green) were not affected (C). (A, B) Phagocytosis of IgG-3-μm beads was reduced by immobilizing lysosomes at the MTOC, n = 80-564 cells; ***P<0.001.


Activation of dynamin - 2 with Ryngo 1 - 23 or expression of constitutively active dynamin - 2 ( S764A mutant , expression confirmed by grayscale image ) restored phagocytosis of IgG - 3 - μm beads ( n = 34 - 51 ) , but had no effect on phagocytosis of IgG - 6 - μm beads , n = 36 - 72 ( E ) ; * * * P < 0 . 001 EtOH Ctrl vs rapalog Ctrl , # # # P < 0 . 001 rapalog Ctrl vs rapalog Ryngo / Dyn2S764A .

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Activation of dynamin-2 with Ryngo 1-23 or expression of constitutively active dynamin-2 (S764A mutant, expression confirmed by the grayscale image) restored phagocytosis of IgG-3-μm beads (n = 34-51 cells), but had no effect on the phagocytosis of IgG-6-μm beads, n = 36-72 cells (E); ***P<0.001 EtOH Ctrl vs rapalog Ctrl, ###P<0.001 rapalog Ctrl vs rapalog Ryngo/Dyn2S764A.


( a ) Relative CK2α over time following microinjection of REF52 with HA - tagged semisynthetic bearing different modifications using immunocytochemistry with HA - specific antibody . Data represent mean values ± s . d . ( b ) Relative CK2α following injection of β subunit together with CK2α . Data represent mean values ± s . d . ( c ) Relative CK2α following injection of proteasome inhibitor ( 1 μM MG132 ) together with unmodified CK2α . Data represent mean values ± s . d .

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(a) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA-tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA-specific antibody. Data represent mean values ± s.d. (b) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins. Data represent mean values ± s.d. (c) Relative CK2α protein concentration following injection of proteasome inhibitor (1 μM MG132) together with unmodified CK2α protein. Data represent mean values ± s.d.


( d ) Western blot for total CK2α in HeLa following treatment with Cdk inhibitors . Numbers indicate relative normalized for loading control . Mean values ( n = 3 ) shown ± s . d .

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(d) Western blot for total CK2α protein in HeLa cells following treatment with Cdk inhibitors. Numbers indicate relative quantification normalized for loading control. Mean values (n = 3) shown ± s.d.


( e ) Western blot for total CK2α in HeLa following treatment with nocodazole to arrest in mitosis . Full gels corresponding to those in panels d e are in Supplementary Figure 8 .

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(e) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis. Full gels corresponding to those in panels d and e are in Supplementary Figure 8.


( a ) Pull - down experiments with semisynthetic HA - CK2α recombinant Pin1 . CK2α was immobilized using HA - specific ( anti - HA ) antibodies bound to G beads . Pin1 was incubated with immobilized CK2α for 20 min at 4 ° C .

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(a) Pull-down experiments with semisynthetic HA-CK2α protein and recombinant Pin1. CK2α was immobilized using HA-specific (anti-HA) antibodies bound to protein G beads. Pin1 was incubated with immobilized CK2α for 20 min at 4 °C.


( b ) Coimmunoprecipitation of endogenous Pin1 with CK2α . Pfa - containing unmodified semisynthetic HA - CK2α were spiked into REF52 lysates immobilized using HA - specific antibodies . Input samples are shown for loading control .

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(b) Coimmunoprecipitation of endogenous Pin1 with CK2α. Pfa-containing and unmodified semisynthetic HA-CK2α proteins were spiked into REF52 cell lysates and immobilized using HA-specific antibodies. Input samples are shown for loading control.


( c ) Pull - down experiments with Pfa344 HA - CK2α recombinant Pin1 in presence of Pin1 - specific or nonspecific IgY .

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(c) Pull-down experiments with Pfa344 HA-CK2α protein and recombinant Pin1 in the presence of Pin1-specific or nonspecific IgY.


( d ) Relative CK2α over time following microinjection of Pfa344 CK2α with Pin1 - specific IgY or nonspecific IgY ( control ) . Data represent mean values ± s . d . Full gels corresponding to those in panels a - c are in Supplementary Figures 10 11 .

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(d) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1-specific IgY or nonspecific IgY (control). Data represent mean values ± s.d. Full gels corresponding to those in panels a-c are in Supplementary Figures 10 and 11.


( a ) Phosphorylation of C - terminal tail CK2α peptides ( residues 337 - 352 ) by Cdk1 - cyclin B . In vitro Cdk1 - cyclin B kinase assays were performed using following peptide substrate : SSMPGGSTPVXSANMMK ( εBiotin ) , where X = serine or S - GlcNAc serine . After reactions were quenched , peptides were separated from reaction mixture on Tris - tricine gels . Data represent mean values ± s . d .

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(a) Phosphorylation of C-terminal tail CK2α peptides (residues 337-352) by Cdk1-cyclin B. In vitro Cdk1-cyclin B kinase assays were performed using the following peptide substrate: SSMPGGSTPVXSANMMK(εBiotin), where X = serine or S-GlcNAc serine. After reactions were quenched, peptides were separated from the reaction mixture on Tris-tricine gels. Data represent mean values ± s.d.


( b ) Immunoprecipitation western blot for O - GlcNAc CK2α in HeLa after treatment with O - GlcNAcase inhibitor TMG for 12 h . Numbers indicate relative normalized for total CK2α concentrations .

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(b) Immunoprecipitation and western blot for O-GlcNAc CK2α in HeLa cells after treatment with the O-GlcNAcase inhibitor TMG for 12 h. Numbers indicate relative quantification normalized for total CK2α concentrations.


( c ) Western blot for total CK2α in HeLa following treatment with TMG for 12 h or 16 h . Numbers indicate relative normalized for loading control , mean values ( n = 4 ) shown ± s . d . Full gels corresponding to those in panels b c are in Supplementary Figures 14 15 .

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(c) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h. Numbers indicate relative quantification normalized for loading control, mean values (n = 4) shown ± s.d. Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15.


Phosphorimage of in vitro CK2 kinase assays with substrates . reactions were performed at 30 ° C . Numbers indicate ( nM ) of product for each kinase reaction . ( a ) AHCYL2 substrate ( 25 ng μl − 1 ) with 100 μM ATP 10 nM CK2α ± 11 nM CK2β for 6 min . ( b ) NAP1L3 substrate ( 15 ng μl − 1 ) with 100 μM ATP 10 nM CK2α ± 11 nM CK2β for 6 min . ( c ) NIPBL substrate ( 50 ng μl − 1 ) with 100 μM ATP 5 nM CK2α ± 11 nM Pin1 for 5 min . Full gels corresponding to those in panels a - c are in Supplementary Figures 19 25 .

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Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates. All reactions were performed at 30 °C. Numbers indicate the concentration (nM) of product for each kinase reaction. (a) AHCYL2 substrate (25 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (b) NAP1L3 substrate (15 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (c) NIPBL substrate (50 ng μl−1) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min. Full gels corresponding to those in panels a-c are in Supplementary Figures 19 and 25.


( B ) of PIN2 phosphopeptide in our experiments . Bars represent mean plus standard error of replicates ( 2 biological replicates for nitrate treatments at 20 min ( Col - 0 roots ) 3 biological replicates for other experimental conditions ) . Each independent biological replicate consisted of a pool of 4 . 500 roots collected from Arabidopsis grown independently under same experimental conditions . asterisk indicates statistically significant differences in phosphoproteomic ( multiple t - comparison without testing corrections , assuming same variance , p < 0 . 05 ) .

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(B) Levels of PIN2 phosphopeptide in our experiments. Bars represent the mean plus standard error of replicates (2 biological replicates for nitrate treatments at 20 min (Col-0 roots) and 3 biological replicates for all other experimental conditions). Each independent biological replicate consisted of a pool of 4.500 roots collected from Arabidopsis plants grown independently under the same experimental conditions. The asterisk indicates statistically significant differences in phosphoproteomic analysis (multiple t-test comparison without testing corrections, assuming same variance, p < 0.05).


( C ) Detection of phosphorylation PIN2 by Phos - tag Western blotting . Arabidopsis ( Col - 0 ) were growth in ammonium as only nitrogen source , treated with 5 mM KNO3 or 5mM KCl as control . Total from roots were analyzed in SDS PAGE using Phos - tag to detect changes in phosphorylation status . Immunoblotting was performed with PIN2 antibody . Total isolated from eir1 . 1 roots were used as a negative control . red asterisks indicate a slow - or fast - mobility band corresponding to a more or less phosphorylated PIN2 , respectively .

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(C) Detection of phosphorylation PIN2 by Phos-tag Western blotting. Arabidopsis plants (Col-0) were growth in ammonium as only nitrogen source, and treated with 5 mM KNO3 or 5mM KCl as control. Total protein from roots were analyzed in SDS PAGE using Phos-tag to detect changes in phosphorylation status. Immunoblotting was performed with PIN2 antibody. Total proteins isolated from eir1.1 roots were used as a negative control. White and red asterisks indicate a slow- or fast-mobility band corresponding to a more or less phosphorylated PIN2, respectively.


( D ) Western blot against PIN2 comparing nitrate treated ( KNO3 ) control ( KCl ) condition in Arabidopsis roots for genotypes ( eir1 - 1 mutant background was complemented with PIN2 : : PIN2wt - GFP ( PIN2wt ) , PIN2 : : PIN2S439D - GFP ( PIN2S439D phospho - mimic point mutation ) or PIN2 : : PIN2S439A - GFP ( PIN2S439A , phospho - null point mutation ) .

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(D) Western blot against PIN2 protein comparing nitrate treated (KNO3) and control (KCl) condition in Arabidopsis roots for all genotypes (eir1-1 mutant background was complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation).


( A ) root of Col - 0 wild type or eir1 - 1 mutant was measured using ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicates statistically significant difference between analyzed by unpaired , two - assuming equal variance t - ( * * p < 0 . 01 ) .

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(A) Primary root length of Col-0 wild type plants or eir1-1 mutant plants was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).


( B ) Number of initiating emerging lateral roots of Col - 0 or eir1 - 1 mutant were counted using microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Asterisk indicates statistically significant difference between analyzed by unpaired , two - assuming equal variance t - ( * * p < 0 . 01 ) .

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(B) Number of initiating and emerging lateral roots of Col-0 or eir1-1 mutant plants were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).


( A ) root of different genotypes was measured using ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - assuming equal variance t - ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(A) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).


( B ) Number of initiating emerging lateral roots for genotypes were counted using microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - assuming equal variance t - ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(B) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).


( A ) Confocal microscopy using Zeiss Airyscan microscope Zeiss - blue3 . 1 software was used to visualize PIN2 in Arabidopsis roots . " e " denotes epidermis " c " cortex . Scale bar = 20 µm . ( B - D ) Tuckey box plots show PIN2 - GFP ( number of roots analyzed per experimental conditions = 8 - 10 ; arbitrary units , a . u . ) at total in roots ( B ) , from epidermal ( C ) cortex ( D ) plasma in different genotypes . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Letters denote statistically significant difference between means as determined by one - way ANOVA followed by Tukey HSD post hoc ( p < 0 . 05 ) .

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(A) Confocal microscopy using Zeiss Airyscan microscope and Zeiss-blue3.1 software was used to visualize PIN2 in Arabidopsis roots. "e" denotes epidermis and "c" cortex. Scale bar = 20 µm. (B-D) Tuckey box plots show PIN2-GFP fluorescence intensity quantification (number of roots analyzed per experimental conditions = 8-10; arbitrary units, a.u.) at the total cell membrane in roots (B), and from epidermal (C) and cortex (D) plasma membrane in the different genotypes. The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Letters denote statistically significant difference between means as determined by one-way ANOVA analysis followed by Tukey HSD post hoc test (p < 0.05).