Figure 2

Trichostatin A increases acetylation and protein stability of p300. (a) T47D cells were treated with or without 1 μM trichostatin A (TSA) for 1 hour. Then 500 μg whole-cell lysate was immunoprecipitated (IP) using anti-p300 or anti-lysine antibody, and western blot (WB) analyzed by anti-p300 or anti-lysine antibody, respectively. The expression of p300 or anti-ERα was analyzed by WB using anti-p300 or anti-ERα antibodies as input. (b) T47D cells were treated with the indicated concentrations of TSA for 3 hours (upper) treated with 1 μM TSA for indicated period (lower). The protein and mRNA expression of p300 was analyzed by WB and RT-PCR, respectively. (c) HeLa cells transiently transfected with 4 μg Myc-ERα were treated with or without 1 μM TSA for 3 hours. Then 500 μg whole-cell lysate was immunoprecipitated using anti-acetylated-lysine (Ac-Lys) or normal IgG antibodies, and then probed by anti-p300 antibody. The expression of p300 and ERα was analyzed by WB using anti-p300 and anti-ERα antibodies as input. (d) HeLa cells were transiently transfected with 4 μg Myc-ERα and treated with various concentrations of TSA for 3 hours. Then 500 μg whole-cell lysate was immunoprecipitated using anti-Ac-Lys antibody, and probed by anti-p300 antibody. The expression of p300 was analyzed by WB using anti-p300 antibodies as input. (e) HeLa cells were transiently transfected with 4 μg Myc-ERα and then treated with 1 μM TSA for 3 hours and 10 μM MG132 for 2 hours as indicated. Then 1 mg whole-cell lysate was immunoprecipitated using anti-Ac-Lys antibody, and probed by p300 antibody. The expression of p300 was analyzed by WB as input. (f) T47D cells were treated with or without 1 μM TSA for 3 hours in the presence of 10 μM cycloheximide (CHX) for the indicated time periods. At the end of treatment, whole-cell lysates were prepared and the expression of proteins analyzed by WB. The density of p300 protein band was determined using an image analysis system. The values were normalized to that of α-tubulin and expressed as a percentage of CHX-untreated control. Each data point represents the mean ± standard error of the mean of at least three independent experiments. **P < 0.01, -TSA vs. +TSA.