p300 increases protein stability of ERα. (a) HeLa cells were transiently transfected with 2 μg Myc-ERα together with 2 μg Myc-p300 or empty vector. At 24 hours after transfection, cells were treated with 20 μM cycloheximide (CHX) for the indicated time periods. The expression of protein was analyzed by western blot (WB) using anti-Myc antibody (upper). The density of 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 (lower). Each data point represents the mean ± standard error of the mean of three independent experiments. **P < 0.01, -p300 vs. +p300. (b) HeLa cells were transiently transfected with 4 μg Myc-ERα together with 2 μg Myc-p300 or empty vector. At 24 hours after transfection, cells were treated with 10 μM MG132 for 2 hours. Then 500 μg whole-cell lysate was immunoprecipitated (IP) using anti-ubiquitin (Ub) or anti-Myc antibody and probed by anti-Myc or anti-Ub antibody, respectively. The expression of ERα was analyzed by WB using anti-Myc antibody as input.