Figure 4
From: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism
SIRT5 is responsible for STAT3 deacetylation in mitochondria.
(a) In HeLa cells SIRT3, SIRT4, and SIRT5 were depleted with siRNA. Immunoprecipitated STAT3 was detected with anti-pan-acetyl-lysine antibody via western blotting. (b) A549 cells were transfected with control or SIRT5 shRNA. Elevated STAT3 acetylation was detected in cytoplasmic, mitochondrial, and nuclear fractions prepared from these cells. Tubulin, porin, and histone H3 were used as cytoplasmic, mitochondrial, and nuclear controls, respectively. (c) In 293T cells, STAT3 full length, 1–130, and 465–770 were cotransfected with Flag-SIRT5. STAT3 association was detected in Flag-SIRT5 immunoprecipitates via western blotting. (d) In 293T cells, Flag-STAT3 was cotransfected with an EV, CBP, CBP and SIRT3 or CBP and SIRT5. Acetylation of immunoprecipitated STAT3 was then detected with a pan-acetyl-lysine antibody. (e) In MEFs, STAT3 was cotransfected with Flag-SIRT5 WT or Flag-SIRT5 H158Y mutant, then subjected to insulin treatment for 30 min. STAT3 acetylation or phosphorylation was detected with the indicated antibodies. (f) A synthetic acetyl-peptide covering the STAT3 K685 site was incubated with Flag-SIRT5 or Flag-SIRT3 protein purified from 293T cells and NAD. Mass spectrometry analysis was then performed. (g) MEFs were transiently transfected with SIRT3 or SIRT5. STAT3 C-terminal acetylation induced by insulin was analysed with specific antibodies against STAT3 acetylation by western blotting, as indicated. SIRT3 and SIRT5 were blotted with anti-Flag; STAT3 was detected in the input.
