( 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 .

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(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 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 .

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(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 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 ) .

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(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 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 . ) .

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(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 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 . ) .

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(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 , 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 . ) .

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(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 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 .

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(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 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 .

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

(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 assay of TAK1 as described in methods .

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


( 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 . ) .

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(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 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 .

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(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 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 .

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(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 the USP4 binding domain of p38IP .

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(H) Mapping the USP4 binding domain of p38IP.


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 .

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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 ) 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 ) .

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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 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 .

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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 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 .

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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.


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 .

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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 , 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 .

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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 ) , 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 .

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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 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 .

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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.


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 ) .

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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 the presence ( BSA - NEFA ) or absence ( BSA ) of extracellular fatty acids ( 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 cells ( 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).


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 .

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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.


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 .

O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

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.


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 ) .

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 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

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).


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 .

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

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 ) 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 .

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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.


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 ) .

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

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 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 ) .

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

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 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 .

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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 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 .

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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 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 .

O O B-MEASURED_VAR 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 B-CONTROLLED_VAR 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

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 the left border primer of the T - DNA insertion . LP and RP represent the left and 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 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 .

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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 The 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 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 .

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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 The 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 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 .

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

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 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 .

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

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 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 .

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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 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 .

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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 and 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 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 .

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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.


Flowering time phenotype analysis for Col - 0 , sec - 5 and 35S : : ATX1 - HA transgenic lines Experiments were repeated three times , for each line , a total of 62 plants was counted for statistical analysis of rosette leaf numbers Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 . n . s . , not significant .

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Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of rosette leaf numbers Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.


Flowering time phenotype analysis for Col - 0 , sec - 5 and 35S : : ATX1 - HA transgenic lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination , respectively . Experiments were repeated three times , for each line , a total of 62 plants was counted for statistical analysis of visible buds percent and bolting rates . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 . n . s . , not significant .

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

Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of visible buds percent and bolting rates. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.


A Detection and conformation of O - GlcNAc modification of His - ATX1ΔN catalyzed by His - SECΔN in vitro . His - ATX1ΔN ( expressing residues 592 - 952 of the C - terminus ) and His - SECΔN ( expressing residues 801 - 1062 of the C - terminus ) were recombinantly expressed and affinity purified separately . O - GlcNAcylation of His - ATX1ΔN was detected by anti - CTD110 . 6 antibody , and further conformed by β - elimination analysis .

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A Detection and conformation of O-GlcNAc modification of His-ATX1ΔN catalyzed by His-SECΔN in vitro. His-ATX1ΔN (expressing residues 592-952 of the C-terminus) and His-SECΔN (expressing residues 801-1062 of the C-terminus) were recombinantly expressed and affinity purified separately. O-GlcNAcylation of His-ATX1ΔN was detected by anti-CTD110.6 antibody, and further conformed by β-elimination analysis.


B His - SECΔN activates His - ATX1ΔN in vitro . Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN . H3K4A : mutated H3 in which the fourth amino acid , a lysine ( K ) , was replaced with alanine ( A ) .

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B His-SECΔN activates His-ATX1ΔN in vitro. Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN. H3K4A: mutated H3 in which the fourth amino acid, a lysine (K), was replaced with alanine (A).


C Analysis of ATX1 O - GlcNAc modification in wild - type and sec - 5 plants . Total soluble protein extracts from 12 - d - old seedlings were subjected to SDS - PAGE followed by immunoblotting using the indicated antibodies . CBB : Coomassie brilliant blue staining , showing relative protein loading amount .

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C Analysis of ATX1 O-GlcNAc modification in wild-type and sec-5 plants. Total soluble protein extracts from 12-d-old seedlings were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. CBB: Coomassie brilliant blue staining, showing relative protein loading amount.


D Loss of SEC function reduced ATX1 activity in Arabidopsis . Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti - ATX1 antibody from wild - type and sec - 5 mutant plants , respectively , and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate . Band intensities were quantified with Image J . The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization .

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D Loss of SEC function reduced ATX1 activity in Arabidopsis. Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti-ATX1 antibody from wild-type and sec-5 mutant plants, respectively, and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate. Band intensities were quantified with Image J. The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization.


E Comparison of ATX1 protein levels in Col - 0 and sec - 5 plants at stages before and after flowering . Seedlings were collected for protein extraction at 21 days ( before flowering ) and 35 days ( after flowering ) after seeds were planted on plates . Anti - ATX1 antibody was used for immunoblot assay .

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E Comparison of ATX1 protein levels in Col-0 and sec-5 plants at stages before and after flowering. Seedlings were collected for protein extraction at 21 days (before flowering) and 35 days (after flowering) after seeds were planted on plates. Anti-ATX1 antibody was used for immunoblot assay.


A Mutation of either S947 alone or all 12 serine and threonine residues in the SET domain reduced O - GlcNAc modification level of ATX1ΔN .

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A Mutation of either S947 alone or all 12 serine and threonine residues in the SET domain reduced O-GlcNAc modification level of ATX1ΔN.


B Site mutation of S947 or all 12 serine and threonine residues in the ATX1 SET domain inhibited the activation of ATX1 by SEC ; moreover , mutation of five conserved amino acids in the SEC functional domain inhibited SEC activity .

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B Site mutation of S947 or all 12 serine and threonine residues in the ATX1 SET domain inhibited the activation of ATX1 by SEC; moreover, mutation of five conserved amino acids in the SEC functional domain inhibited SEC activity.


C Quantitative analysis of HKMT activity of ATX1ΔN , ATX1ΔN - 12m , and ATX1ΔN - m and the effect of SECΔN and ΔSEC5m in activating those proteins . Experiments were repeated three times . Error bars are SD . His - ΔSEC5m and His - SECΔN were used as the control .

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C Quantitative analysis of HKMT activity of ATX1ΔN, ATX1ΔN-12m, and ATX1ΔN-m and the effect of SECΔN and ΔSEC5m in activating those proteins. Experiments were repeated three times. Error bars are SD. His-ΔSEC5m and His-SECΔN were used as the control.


A Flowering phenotypes of Col - 0 , atx1 - 2 , atx1 - 2 35S : : ATX1 - FLAG , and atx1 - 2 35S : : ATX1m - FLAG ( with Ser947 replaced with alanine ) plants under LD conditions . Scale bar : 1 cm . Two independent lines are shown for each transformation .

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A Flowering phenotypes of Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG (with Ser947 replaced with alanine) plants under LD conditions. Scale bar: 1 cm. Two independent lines are shown for each transformation.


B Quantitative real - time PCR analysis of ATX1 and FLC transcript levels in Col - 0 , atx1 - 2 , atx1 - 2 35S : : ATX1 - FLAG , and atx1 - 2 35S : : ATX1m - FLAG plants . The transcript levels were normalized to that of TUBULIN . Data are mean ± s . d . of three independent biological replicates , n = 3 .

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B Quantitative real-time PCR analysis of ATX1 and FLC transcript levels in Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG plants. The transcript levels were normalized to that of TUBULIN. Data are mean ± s.d. of three independent biological replicates, n=3.


Flowering time phenotype analysis of lines Three independent biological repeats were conducted , and data from a total of 71 plants were used for statistical analysis . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 .

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Flowering time phenotype analysis of lines Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01.


Flowering time phenotype analysis of lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination , respectively . Three independent biological repeats were conducted , and data from a total of 71 plants were used for statistical analysis . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 .

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Flowering time phenotype analysis of lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01.


B : U2 - OS cells were transfected with the indicated siRNAs . The indicated cells were treated with etoposide ( 50 µM , 90 min . before analysis ) . Whole - cell extracts were immunoblotted with the indicated antibodies . The result is representative of three biological replicates .

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B: U2-OS cells were transfected with the indicated siRNAs. The indicated cells were treated with etoposide (50 µM, 90 min. before analysis). Whole-cell extracts were immunoblotted with the indicated antibodies. The result is representative of three biological replicates.


D : U2 - OS cells were transfected with non - targeting ( mock ) or TSG101 - targeting siRNAs and irradiated ( 20 Gy ) 90 min . before analysis . Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one - way ANOVA ( * p < 0 . 05 ) in the right panel .

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D: U2-OS cells were transfected with non-targeting (mock) or TSG101-targeting siRNAs and irradiated (20 Gy) 90 min. before analysis. Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one-way ANOVA (*p < 0.05) in the right panel.


A : Nuclear - cytoplasmic fractionation of U2 - OS cells . Indicated samples were transfected with siRNAs against TSG101 . PARylation was inhibited by olaparib ( 10 µM , 24 hours before analysis ) . Cytoplasmic export of p - ATM was induced by etoposide ( 50 µM , 45 minutes before analysis ) . Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies . The fractionation efficiency was controlled by the respective subcellular marker proteins ( PARP1 and LDH - A ) . PARylation was detected using the Pan ADPr reagent . Note that PARylation is activated by shearing forces during extract preparation ( lane 1 )

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A: Nuclear-cytoplasmic fractionation of U2-OS cells. Indicated samples were transfected with siRNAs against TSG101. PARylation was inhibited by olaparib (10 µM, 24 hours before analysis). Cytoplasmic export of p-ATM was induced by etoposide (50 µM, 45 minutes before analysis). Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. The fractionation efficiency was controlled by the respective subcellular marker proteins (PARP1 and LDH-A). PARylation was detected using the Pan ADPr reagent. Note that PARylation is activated by shearing forces during extract preparation (lane 1)


B : U2 - OS cells were transfected with non - targeting ( - ) or TSG101 - targeting ( + ) siRNAs and cells were irradiated ( + ) or not ( - ) as indicated ( 20 Gy ) 10 min . before analysis . Whole - cell extracts were immunoblotted with the 10H PAR monoclonal antibody .

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B: U2-OS cells were transfected with non-targeting (-) or TSG101-targeting (+) siRNAs and cells were irradiated (+) or not (-) as indicated (20 Gy) 10 min. before analysis. Whole-cell extracts were immunoblotted with the 10H PAR monoclonal antibody.


C : siRNA - transfected U2 - OS cells as in B were etoposide - treated or not , as indicated . Whole - cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly - and mono - ( ADP - ribose ) . Note that PARylation also occurred during cell lysis in untreated cells .

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C: siRNA-transfected U2-OS cells as in B were etoposide-treated or not, as indicated. Whole-cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly- and mono-(ADP-ribose). Note that PARylation also occurred during cell lysis in untreated cells.


D : U2 - OS cells were transfected with non - targeting or VPS28 - targeting siRNAs . Whole - cell extracts were immunoblotted with indicated antibodies .

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D: U2-OS cells were transfected with non-targeting or VPS28-targeting siRNAs. Whole-cell extracts were immunoblotted with indicated antibodies.


F : Cells were irradiated ( 20 Gy ) as indicated 10 min before analysis . Whole - cell extracts were immunoblotted with the indicated antibodies .

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F: Cells were irradiated (20 Gy) as indicated 10 min before analysis. Whole-cell extracts were immunoblotted with the indicated antibodies.


A : Proximity ligation assay ( PLA ) assay with unstimulated , etoposide ( Eto ) - treated ( 50 µM , 10 min . before analysis ) , or etoposide plus olaparib - ( Olap ) co - treated ( 50 µM of etoposide , 10 min . before analysis and 10 µM of olaparib 24 hours before analysis ) U2 - OS cells . The red dots represent PARP1 - TSG101 interaction . The nucleus is stained with DAPI ( blue ) . Scale bar : 10 µm . As negative controls , PLA was performed without primary PARP1 or TSG101 antibodies . As an additional control , PLA was performed in PARP1 depleted cells to show specificity B : Quantification of PLA dots per nuclei in A . Blind counting was performed from 30 replicates three biologically independent experiments . The conditions were compared with an ordinary one - way ANOVA ( ns , p > 0 . 05 ; * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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A: Proximity ligation assay (PLA) assay with unstimulated, etoposide (Eto)-treated (50 µM, 10 min. before analysis), or etoposide plus olaparib-(Olap) co-treated (50 µM of etoposide, 10 min. before analysis and 10 µM of olaparib 24 hours before analysis) U2-OS cells. The red dots represent PARP1-TSG101 interaction. The nucleus is stained with DAPI (blue). Scale bar: 10 µm. As negative controls, PLA was performed without primary PARP1 or TSG101 antibodies. As an additional control, PLA was performed in PARP1 depleted cells to show specificity B: Quantification of PLA dots per nuclei in A. Blind counting was performed from 30 replicates three biologically independent experiments. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001).


C : U2 - OS cells were cotransfected with the indicated deletion constructs of mCherry - tagged TSG101 and EGFP - tagged PARP1 . Immunoprecipitation of GFP was performed using whole - cell extracts .

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C: U2-OS cells were cotransfected with the indicated deletion constructs of mCherry-tagged TSG101 and EGFP-tagged PARP1. Immunoprecipitation of GFP was performed using whole-cell extracts.


F : In - vitro PARylation reactions were carried out with the indicated recombinant and purified proteins , including 8 ng PARP1 in the presence of sssDNA ( sheared salmon sperm DNA ) and MgCl2 . 100 ng of TSG101 was added to the reaction , as indicated . The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls .

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F: In-vitro PARylation reactions were carried out with the indicated recombinant and purified proteins, including 8 ng PARP1 in the presence of sssDNA (sheared salmon sperm DNA) and MgCl2. 100 ng of TSG101 was added to the reaction, as indicated. The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls.


B : Representative images of PARP1 - GFP association with laser - microirradiation sites in untreated ( mock ) , olaparib - treated ( 10 µM , 24 hours before analysis ) or siTSG101 - transfected U2 - OS cells at indicated times post - irradiation . Scale bar : 7 . 5 µm . The image is representative of nine replicates from three biologically independent experiments . Go to Movies EV1 - 3 for further illustration .

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B: Representative images of PARP1-GFP association with laser-microirradiation sites in untreated (mock), olaparib-treated (10 µM, 24 hours before analysis) or siTSG101-transfected U2-OS cells at indicated times post-irradiation. Scale bar: 7.5 µm. The image is representative of nine replicates from three biologically independent experiments. Go to Movies EV1-3 for further illustration.


C : Kinetics of PARP1 - GFP recruitment and dissociation from DNA lesions were measured at times indicated . Nine nuclei were analyzed for each indicated condition from three biologically independent experiments . The data are shown as mean GFP intensity in the microirradiated area ± SEM , normalized to the mean GFP intensity of corresponding whole nuclei . D : Relative PARP1 - GFP recruitment to DNA lesions for each indicated condition at 12 min . post - irradiation .

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C: Kinetics of PARP1-GFP recruitment and dissociation from DNA lesions were measured at times indicated. Nine nuclei were analyzed for each indicated condition from three biologically independent experiments. The data are shown as mean GFP intensity in the microirradiated area ± SEM, normalized to the mean GFP intensity of corresponding whole nuclei. D: Relative PARP1-GFP recruitment to DNA lesions for each indicated condition at 12 min. post-irradiation.


C : Cells were transfected with non - targeting or TSG101 - targeting siRNAs . Immunofluorescence staining was performed with the indicated antibodies ( red : cleaved caspase 3 ; green : γH2AX ) . Nuclei were stained with DAPI ( blue ) . The image is a representative of 3 biologically independent experiments . Scale bar : 30 µm . See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR - treatment . D : Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments . Conditions were compared with an unpaired t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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C: Cells were transfected with non-targeting or TSG101-targeting siRNAs. Immunofluorescence staining was performed with the indicated antibodies (red: cleaved caspase 3; green: γH2AX). Nuclei were stained with DAPI (blue). The image is a representative of 3 biologically independent experiments. Scale bar: 30 µm. See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR-treatment. D: Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. Conditions were compared with an unpaired t-test (*p < 0.05, **p < 0.01).


E : γH2AX staining of TSG101 - targeting or non - targeting siRNA transfected U2 - OS cells reveals puncta - like foci at 2 or 7 days after irradiation .

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E: γH2AX staining of TSG101-targeting or non-targeting siRNA transfected U2-OS cells reveals puncta-like foci at 2 or 7 days after irradiation.


F : The number of puncta - like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments .

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F: The number of puncta-like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments.


Representative images of seed fluorescence segregation in 420 / + + in wild type ( Col ) and hcr2 . Scatterplots to the right show red ( dsRed ) and green ( eGFP ) fluorescence values in 420 / + + Col ( top ) and hcr2 ( bottom ) . Scale bars : 2 mm .

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Representative images of seed fluorescence segregation in 420/++ in wild type (Col) and hcr2. Scatterplots to the right show red (dsRed) and green (eGFP) fluorescence values in 420/++ Col (top) and hcr2 (bottom). Scale bars: 2 mm.


End - point RT - PCR analysis of HSBP in Col , hsbp - 3 , hsbp - 2 and hsbp - 1 . Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp - 3 , respectively . Image J was used to measure relative PCR band intensity for hsbp - 3 ( 53 % ) , hsbp - 2 ( 9 % ) and hsbp - 1 ( 70 % ) . TUB2 was used as an internal control . As in E , but showing immunoblot analysis of HSBP . hsbp - 3 , hsbp - 2 and hsbp - 1 accumulate about 58 % , 17 % and 77 % of HSBP levels , respectively . Coomassie - stained membrane was used as a loading control .

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End-point RT-PCR analysis of HSBP in Col, hsbp-3, hsbp-2 and hsbp-1. Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp-3, respectively. Image J was used to measure relative PCR band intensity for hsbp-3 (53%), hsbp-2 (9%) and hsbp-1 (70%). TUB2 was used as an internal control. As in E, but showing immunoblot analysis of HSBP. hsbp-3, hsbp-2 and hsbp-1 accumulate about 58%, 17% and 77% of HSBP levels, respectively. Coomassie-stained membrane was used as a loading control.


Representative images of ASY1 ( green ) and RAD51 ( red ) immunostaining in Col and hsbp - 3 . Nuclei spreads were stained with DAPI ( blue ) . Scale bars : 10 μm . Quantification of RAD51 foci numbers per cell in Col ( blue ) and hsbp - 3 ( red ) . n = 20 cells of biological replicates .

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Representative images of ASY1 (green) and RAD51 (red) immunostaining in Col and hsbp-3. Nuclei spreads were stained with DAPI (blue). Scale bars: 10 μm. Quantification of RAD51 foci numbers per cell in Col (blue) and hsbp-3 (red). n = 20 cells of biological replicates.


Representative images of MLH1 ( red ) immunostaining in Col , hsbp - 3 , hsbp - 2 , Col / Ler , and meiMIGS - HSBP Col / Ler . Nuclear DNA was stained with DAPI . Scale bar : 5 μm . Quantification of the number of MLH1 foci per cell shown in ( G ) . n ≥ 32 cells of biological replicates .

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Representative images of MLH1 (red) immunostaining in Col, hsbp-3, hsbp-2, Col/Ler, and meiMIGS-HSBP Col/Ler. Nuclear DNA was stained with DAPI. Scale bar: 5 μm. Quantification of the number of MLH1 foci per cell shown in (G). n ≥ 32 cells of biological replicates.


Representative images of HEI10 ( red ) and ASY1 ( green ) immunostaining in Col and hsbp - 3 . Scale bar : 2 μm .

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Representative images of HEI10 (red) and ASY1 (green) immunostaining in Col and hsbp-3. Scale bar: 2 μm.


Quantification of the number of HEI10 foci per cell shown in ( I ) . n ≥ 21 cells of biological replicates .

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Quantification of the number of HEI10 foci per cell shown in (I). n ≥ 21 cells of biological replicates.


Immunoblot analysis of HEI10 and HEI10 - Myc in Col , hsbp - 3 , HEI10 - myc and HEI10 - myc plants . Experiments were performed at least three times .

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Immunoblot analysis of HEI10 and HEI10-Myc in Col, hsbp-3, HEI10-myc and HEI10-myc plants. Experiments were performed at least three times.


Figure 1 . DENND2B binds ITSN Purified GST - SH3 - containing proteins were incubated with lysates of HEK - 293T cells expressing Flag - DENND2B . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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Figure 1. DENND2B binds ITSN  Purified GST-SH3-containing proteins were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Purified GST - SH3 domains of ITSN were incubated with lysates of HEK - 293T cells expressing Flag - DENND2B . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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Purified GST-SH3 domains of ITSN were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Lysates from HEK - 293T cells expressing Flag - ITSN - s and GFP or GFP - DENND2B were incubated with protein A beads or protein A beads coupled to anti - GFP . Bound proteins were detected by Western blot .

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Lysates from HEK-293T cells expressing Flag-ITSN-s and GFP or GFP-DENND2B were incubated with protein A beads or protein A beads coupled to anti-GFP. Bound proteins were detected by Western blot.


D , E HeLa cells expressing the indicated constructs . Flag - ITSN - s was visualized with anti - Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively . Scale bar : 5 µm . Boxed regions are magnified on the bottom . White arrows and red arrows point to colocalizing and non - colocalizing structures , respectively .

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D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.


Figure 2 . DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK - 293T cells were treated with DMSO or okadaic acid ( OA ) , lysed and analyzed by Western blot .

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Figure 2. DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK-293T cells were treated with DMSO or okadaic acid (OA), lysed and analyzed by Western blot.


B HEK - 293T cells expressing Flag - DENND2B were treated with DMSO or 250 nM OA , and cell lysates were incubated with GST - proteins . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST-proteins. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry . Low abundance peptides not included . Refer to Table EV1 for a complete peptide list .

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C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry. Low abundance peptides not included. Refer to Table EV1 for a complete peptide list.


E HEK - 293T cells expressing Flag - DENND2B were treated with DMSO , 250 nM OA or with increasing concentrations of PKD inhibitor CID755673 . Cell lysates were analyzed by Western blot .

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E HEK-293T cells expressing Flag-DENND2B were treated with DMSO, 250 nM OA or with increasing concentrations of PKD inhibitor CID755673. Cell lysates were analyzed by Western blot.


F HEK - 293T cells expressing Flag - DENND2B or control cells were treated with 250 nM OA or DMSO , incubated with protein G beads coupled to anti - Flag and analyzed by Western blot .

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F HEK-293T cells expressing Flag-DENND2B or control cells were treated with 250 nM OA or DMSO, incubated with protein G beads coupled to anti-Flag and analyzed by Western blot.


G Lysates from HEK - 293T cells expressing Flag - DENND2B and PKD - dominant negative ( DN ) were incubated with protein G beads or protein G beads coupled to anti - Flag . Bound proteins were detected by Western blot .

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G Lysates from HEK-293T cells expressing Flag-DENND2B and PKD-dominant negative (DN) were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.


H HEK - 293T cells co - expressing Flag - DENND2B with pcDNA3 or PKD - constitutively active ( CA ) , were incubated with protein G beads or protein G beads coupled to anti - Flag and bound proteins were detected by Western blot .

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H HEK-293T cells co-expressing Flag-DENND2B with pcDNA3 or PKD-constitutively active (CA), were incubated with protein G beads or protein G beads coupled to anti-Flag and bound proteins were detected by Western blot.


I Quantification of H where PKD - CA treatment is normalized to pcDNA3 control . Mean + / - SD . Welch ' s t - test * p = 0 . 016 , n = 6 for both treatment groups from 3 independent experiments .

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I Quantification of H where PKD-CA treatment is normalized to pcDNA3 control. Mean +/- SD. Welch's t-test *p=0.016, n=6 for both treatment groups from 3 independent experiments.


Figure 3 . 14 - 3 - 3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST - 14 - 3 - 3 WT or K50E were incubated with lysates of HEK - 293T cells expressing GFP - DENND2B WT or S30A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


B HEK - 293T cells expressing Flag - DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST - 14 - 3 - 3 . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


C Quantification of B . where bound DENND2B is normalized to the SM . Mean + / - SD . Welch ' s t - test * * p = 0 . 006 n = 8 for both treatment groups from 5 independent

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C Quantification of B. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test **p=0.006 n=8 for both treatment groups from 5 independent experiments


D HEK - 293T cells expressing Flag - DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST - 14 - 3 - 3 . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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D HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


E Quantification of D . where bound DENND2B is normalized to the SM . Mean + / - SD . Welch ' s t - test * p = 0 . 040 . n = 5 for both treatment groups from 3 independent

E Quantification of D. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test *p=0.040. n=5 for both treatment groups from 3 independent experiments


F GST - SH3A was incubated with lysates of HEK - 293T cells expressing GFP - DENND2B WT or S30A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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F GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


G Lysates of HEK - 293T cells expressing Flag - DENND2B were pre - incubated with increasing concentrations of purified 14 - 3 - 3 WT and subsequently incubated with GST - SH3A ( top panel ) or GST - Grb2 ( bottom panel ) . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Figure 4 . DENND2B promotes EGFR recycling A , B HeLa cells expressing the indicated constructs . Boxed regions are magnified on the bottom . Scale bars : 5 µm in top panel and 1 µm bottom panel . White arrows and arrowheads point to colocalization on vesicles and plasma membrane , respectively .

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Figure 4. DENND2B promotes EGFR recycling A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.


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C Transduced MCF10A cells were quantified by real - time PCR . Mean + / - SD , ANOVA with Dunnett post - hoc test * * * p = 0 . 000 for both comparisons , n = 3 from 3 independent experiments .

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C Transduced MCF10A cells were quantified by real-time PCR. Mean +/- SD, ANOVA with Dunnett post-hoc test ***p=0.000 for both comparisons, n=3 from 3 independent experiments.


D Transduced HEK - 293T cells expressing Flag - DENND2B were analyzed by Western blot .

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D Transduced HEK-293T cells expressing Flag-DENND2B were analyzed by Western blot.


E Transduced MCF10A cells , were biotinylated and cell lysates were incubated with streptavidin beads . Bound proteins were detected by Western blot .

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E Transduced MCF10A cells, were biotinylated and cell lysates were incubated with streptavidin beads. Bound proteins were detected by Western blot.


F Quantification of E . where surface EGFR is normalized to total EGFR . Mean + / - SD , Anova with Dunnett post - hoc test * p = 0 . 013 and * * * p = 0 . 000 . n = 6 for all treatment groups from 4 independent

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F Quantification of E. where surface EGFR is normalized to total EGFR. Mean +/- SD, Anova with Dunnett post-hoc test *p=0.013 and ***p=0.000. n=6 for all treatment groups from 4 independent experiments


G Quantification of E where total EGFR is normalized to Hsc70 . Mean + / - SD n = 3 for all treatment groups from 3 independent

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G Quantification of E where total EGFR is normalized to Hsc70. Mean +/- SD n=3 for all treatment groups from 3 independent experiments


I MCF10A cells expressing GFP - DENND2B were labelled for surface EGFR on ice , incubated at 16 ° C to allow internalization , acid washed to remove remaining labelled - EGFR and processed for immunofluorescence to visualize the internal pool of EGFR ( post - endocytosis , top panel ) or incubated at 37 ° C to allow recycling , acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR ( post - recycling , bottom panel ) .

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I MCF10A cells expressing GFP-DENND2B were labelled for surface EGFR on ice, incubated at 16°C to allow internalization, acid washed to remove remaining labelled-EGFR and processed for immunofluorescence to visualize the internal pool of EGFR (post-endocytosis, top panel) or incubated at 37°C to allow recycling, acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR (post-recycling, bottom panel).


J Quantification of I top panel , internal EGFR post - endocytosis . Mean fluorescence per cell + / - SD , n = 13 for GFP - DENND2B and n = 21 for control cells pooled from 2 independent experiments .

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J Quantification of I top panel, internal EGFR post-endocytosis. Mean fluorescence per cell +/- SD, n=13 for GFP-DENND2B and n=21 for control cells pooled from 2 independent experiments.


K Quantification of I bottom panel , internal EGFR post - recycling . Mean fluorescence per cell + / - SD , n = 10 for GFP - DENND2B and n = 26 for control cells pooled from 2 independent experiments .

K Quantification of I bottom panel, internal EGFR post-recycling. Mean fluorescence per cell +/- SD, n=10 for GFP-DENND2B and n=26 for control cells pooled from 2 independent experiments.


Figure 5 . EGF treatment dissociates DENND2B from ITSN A HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for the indicated times and cell lysates were analyzed by Western blot .

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Figure 5. EGF treatment dissociates DENND2B from ITSN A HEK-293T cells were serum starved, treated with 100 ng/mL EGF for the indicated times and cell lysates were analyzed by Western blot.


B Quantification of A where P - PKD is normalized to Hsc70 . Mean + / - SD . ANOVA with Dunnett post - test * p = 0 . 015 and * * p = 0 . 003 n = 3 from 3 independent experiments .

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B Quantification of A where P-PKD is normalized to Hsc70. Mean +/- SD. ANOVA with Dunnett post-test *p=0.015 and ** p=0.003 n=3 from 3 independent experiments.


C HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti - Flag . Bound proteins were detected by Western blot .

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C HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.


D Quantification of B where P - PKD substrate is normalized to Flag - DENND2B . Mean + / - SD Welch ' s t - test * p = 0 . 036 , n = 4 from 3 independent experiments .

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D Quantification of B where P-PKD substrate is normalized to Flag-DENND2B. Mean +/- SD Welch's t-test *p=0.036, n=4 from 3 independent experiments.


E HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for 5 min and cell lysates were incubated with GST - SH3A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively .

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E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


F Quantification of E where bound Flag - DENND2B is normalized to SM . Mean + / - SD , Welch ' s t - test * p = 0 . 012 , n = 6 from 3 independent experiments .

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F Quantification of E where bound Flag-DENND2B is normalized to SM. Mean +/- SD, Welch's t-test *p=0.012, n=6 from 3 independent experiments.


( A , B ) In situ cryo - electron tomogram displaying the proximal region of a mature mother centriole ( A ) and procentriole ( B ) . Mature centriole , MC ; procentriole , PC ; mitochondria , mito ; vacuole , vac ; white dashed line , lamella edge . Scale bars , 100 nm .

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(A, B) In situ cryo-electron tomogram displaying the proximal region of a mature mother centriole (A) and procentriole (B). Mature centriole, MC; procentriole, PC; mitochondria, mito; vacuole, vac; white dashed line, lamella edge. Scale bars, 100 nm.


( C ) Side view z - projection of cartwheels containing the central hub and several CIDs from a mature centriole . Central hub , CH ; cartwheel inner densities , CID . Scale bar , 20 nm .

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(C) Side view z-projection of cartwheels containing the central hub and several CIDs from a mature centriole. Central hub, CH; cartwheel inner densities, CID. Scale bar, 20 nm.


( D ) Cross section of the cartwheel - containing regions from a mature centriole . Microtubule triplet , MTT ; spokes , SP . Scale bar , 200 nm .

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(D) Cross section of the cartwheel-containing regions from a mature centriole. Microtubule triplet, MTT; spokes, SP. Scale bar, 200 nm.


( E ) Side view z - projection of cartwheels containing the central hub and several CIDs from a procentriole . Scale bar , 20 nm .

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(E) Side view z-projection of cartwheels containing the central hub and several CIDs from a procentriole. Scale bar, 20 nm.


( F ) Cross section of the cartwheel - containing regions from a procentriole . Scale bar , 200 nm .

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(F) Cross section of the cartwheel-containing regions from a procentriole. Scale bar, 200 nm.


( G ) Nine - fold symmetrized cross sections of the cartwheel - containing region from a mature centriole ( left side ) and a procentriole ( right side ) . Dashed white circle , central hub . Scale bars , 100 nm .

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(G) Nine-fold symmetrized cross sections of the cartwheel-containing region from a mature centriole (left side) and a procentriole (right side). Dashed white circle, central hub. Scale bars, 100 nm.


( H ) Longitudinal periodicity measurements of the central hub and CIDs . Central hub , blue ; CID , red . Mean values are displayed above the data range . Blue data points are measured distance between individual units of the central hub , red data points are measured distances between individual units of the cartwheel inner density . Mature centriole , central hub , n = 30 , mean = 4 . 1 + / - 0 . 67 ( S . D ) ; mature centriole , cartwheel inner density , n = 18 , mean = 8 . 7 + / - 1 . 6 ( S . D ) ; procentriole , central hub , n = 10 , mean = 4 . 0 + / - 0 . 66 ( S . D ) ; procentriole , cartwheel inner density , n = 5 , mean = 8 . 6 + / - 1 . 2 ( S . D ) .

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(H) Longitudinal periodicity measurements of the central hub and CIDs. Central hub, blue; CID, red. Mean values are displayed above the data range. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. Mature centriole, central hub, n = 30, mean = 4.1 +/- 0.67 (S.D); mature centriole, cartwheel inner density, n = 18, mean = 8.7 +/- 1.6 (S.D); procentriole, central hub, n = 10, mean = 4.0 +/- 0.66 (S.D); procentriole, cartwheel inner density, n = 5, mean = 8.6 +/- 1.2 (S.D).


( I , J ) Nine - fold symmetrized central hub z - projections , starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5 . 4 nm steps in a mature centriole ( I ) and a procentriole ( J ) . Red arrow , CID ; blue arrow ; central hub . Scale bar : 20 nm .

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(I, J) Nine-fold symmetrized central hub z-projections, starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5.4 nm steps in a mature centriole (I) and a procentriole (J). Red arrow, CID; blue arrow; central hub. Scale bar: 20 nm.


( A , B , C ) Cryo - electron tomograms of the proximal regions of a P . tetraurelia centriole ( A ) , a N . gruberi centriole ( B ) , and a H . sapiens procentriole ( C ) . White arrows denote a broken cartwheel ; procentriole , PC ; mature centriole , MC ; Scale bar , 100 nm . Note that most N . gruberi and H . sapiens centrioles were heavily compressed during the cryo - EM preparation , as previously described ( Guichard et al . , 2010 ) . The displayed N . gruberi centriole illustrates the damage caused by compression . The periodicities of N . gruberi and H . sapiens cartwheels were measured only on regions that were not

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(A, B, C) Cryo-electron tomograms of the proximal regions of a P. tetraurelia centriole (A), a N. gruberi centriole (B), and a H. sapiens procentriole (C). White arrows denote a broken cartwheel; procentriole, PC; mature centriole, MC; Scale bar, 100 nm. Note that most N. gruberi and H. sapiens centrioles were heavily compressed during the cryo-EM preparation, as previously described (Guichard et al., 2010). The displayed N. gruberi centriole illustrates the damage caused by compression. The periodicities of N. gruberi and H. sapiens cartwheels were measured only on regions that were not damaged


( D ) Cross sections from cartwheel - containing region of P . tetraurelia centrioles . Scale bar , 50 nm . ( E ) Zoomed side view of cartwheel from P . tetraurelia , displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow . Scale bar , 25 nm .

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(D) Cross sections from cartwheel-containing region of P. tetraurelia centrioles. Scale bar, 50 nm. (E) Zoomed side view of cartwheel from P. tetraurelia, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow. Scale bar, 25 nm.


( F ) Cross section from cartwheel - containing region of N . gruberi , ( G ) Zoomed side view of cartwheel from N . gruberi displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow .

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(F) Cross section from cartwheel-containing region of N. gruberi, (G) Zoomed side view of cartwheel from N. gruberi displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow.