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. 2017 Oct 6;25(2):255–267. doi: 10.1038/cdd.2017.152

Figure 6.

Figure 6

GADD34 promotes dephosphorylation of phosphoserine-47 on SIRT1. (a) WT or GADD34−/− MEFs expressing GFP-SIRT1 were treated with 50 μM Ars for 0, 2 and 4 h before immunoblotting for phosphoserine-47 SIRT1 (pSer47-SIRT1), GFP-SIRT1, phosphoserine-51 eIF2α (p-eIF2α), eIF2α, GADD34 and tubulin. (b) Ratio of pSer47-SIRT1/GFP-SIRT1 level in WT or GADD34−/− MEFs treated with Ars for indicated times is shown as a bar graph (mean±S.E.M., n=3 independent experiments). Tuke’s multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05, NS denotes not significant). (c) Fold change of p-eIF2α/eIF2α in WT or GADD34−/− MEFs treated with Ars for indicated times is shown as a bar graph (mean±S.E.M., n=3 independent experiments). Dunnett’s multiple comparisons test was used after two-way ANOVA to generate P-values (*P<0.05, NS denotes not significant). (d) HEK293 cells expressing FLAG empty vector, FLAG-GADD34 or FLAG KARA were either UT (−) or Ars-treated (+)before immunoprecipitations using anti-FLAG conjugated beads. IP and WCLs were immunoblotted for SIRT1, eIF2α, PP1α and the FLAG-GADD34. (e) GFP-SIRT1 was coexpressed with either empty FLAG vector, FLAG-GADD34 or FLAG-KARA in GADD34−/− MEFs. As control, GFP-SIRT1 was co-expressed with empty FLAG vector in WT MEFs. Transfected MEFs were UT (−) or treated with 50 μM Ars for 4 h and immunoblotted as described in a. (f) Ratio of pSer47-SIRT1/GFP-SIRT1 in WT or GADD34−/− MEFs expressing various FLAG plasmids with or without Ars treatment (mean±S.E.M., n=3). Sidak multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05)