Figure 1

Growth factor induces STAT3 acetylation in serum-starved cells.

(a) Time course of induction of STAT3 acetylation by serum starvation and reintroduction (SSR) (left) or serum starvation and insulin treatment (right) in PC3 cells transfected with STAT3. Pan acetyl-lysine antibody was used to detect acetyl-STAT3. (b) Alignment of C-terminal dimerization domain sequences from STAT1, STAT3, and STAT5b. K685, K707, and K709 are acetylation sites identified by mass spectrometry analysis of STAT3 prepared from PC3 cells transfected with STAT3. Y705 is phosphorylated after cytokine treatment. (c) SSR for 30 min (left) or insulin (5 μg/ml) for 15 min (right) induced STAT3 post-translational modifications, as indicated in MEFs that were serum-starved (−) for at least 6 hrs. (d) 293T cells were transfected with STAT3 or STAT3 and CBP, and this was followed by a 6-hr treatment with NAM (100 μM, 200 μM) or TSA (1 μM, 5 μM). Whole cell lysates were prepared and subjected to western blotting with the indicated antibodies. Acetyl-STAT3 was detected with anti-pan acetyl-lysine antibody. (e) CBP was depleted with siRNA in 293T cells. After insulin treatment for 30 min, WCL were prepared, and acetyl-STAT3 was blotted with the anti-pan acetyl-lysine antibody. (f) In 293T cells, CBP was transfected for 36 hrs before harvesting. Cytoplasmic, mitochondrial, and nuclear fractions were prepared for CBP and mitochondrial marker porin analysis with specific antibodies using western blotting. (g) MEFs were treated with insulin for 30 min or were left untreated. CBP was immunoprecipitated and subjected to blotting with anti-insulin receptor or anti-CBP antibodies. (h) In 293T cells, CBP was transfected for 36 hrs before harvest. Cytoplasmic, mitochondrial and nuclear fractions were prepared for STAT3 analysis.