Figure 5.

Inhibition of HDACs by TSA and inhibition of HDAC6 by ACY-1215 increase endogenous ERK1 and ERK2 acetylation. A, TSA increases endogenous ERK1/2 acetylation. Left panels, HEK293T cells were treated with a vehicle or 600 ng/ml TSA for 24 h. The anti-AcK antibody was used to immunoprecipitate (IP) acetylated ERK1/2. The immunoprecipitates were resolved on SDS-PAGE, and the anti-ERK1/2 Western blot analysis was performed. 0.5% of whole-cell lysate was used as the input. Western blot analyses were performed using anti-ERK1/2, anti-acetyl–α-tubulin, or α-tubulin antibodies as indicated. Right panel, bar graph was used to show relative intensity of endogenous ERK1 and ERK2. Three independent experiments were performed. B, ACY-1215 increases endogenous ERK1/2 acetylation. The experiments were performed as described in A except that ACY-1215 was used for the treatment instead of TSA. Four independent experiments were performed. Student's t tests were performed with *, p < 0.05; **, p < 0.01. Error bars, S.D. IB, immunoblot.