Figure 1

Trichostatin A enhances acetylation as well as stability of ERα protein. (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-ERα, anti-acetylated-lysine (Ac-Lys) or normal IgG antibodies, and western blot (WB) analyzed by anti-Ac-Lys or anti-ERα antibody as indicated. The expression of ERα and α-tubulin was analyzed by WB as input. (b) The ERα-positive T47D cells were treated with the indicated concentrations of TSA for 3 hours. The protein and mRNA expression of ERα were analyzed by WB and RT-PCR, respectively. (c) HeLa cells were transiently transfected with 4 μg Myc-ERα and then treated with or without 1 μM TSA for 3 hours. Then 500 μg whole-cell lysate was immunoprecipitated using anti-pan-Ac, anti-Ac-Lys or normal IgG antibodies, and probed by anti-Myc antibody. The expression of ERα was analyzed by WB using anti-Myc or anti-ERα antibodies as input. (d) HeLa cells were transiently transfected with 4 μg Myc-ERα and then treated with various concentrations of TSA for 3 hours. Then 500 μg whole-cell lysate was immunoprecipitated using anti-pan-Ac antibody, and probed by anti-Myc antibody. The expression of ERα was analyzed by WB using anti-Myc or anti-ERα 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-Lys-Ac antibody, and probed by anti-Myc antibody. The expression of ERα was analyzed by WB using anti-Myc antibodies 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 was analyzed by WB. Density of the ERα 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.