Effects of signaling inhibitors on ER-α acetylation due to BRCA1 knockdown. A, Subconfluent proliferating MCF-7 cells were treated with BRCA1 siRNA or control (CON) siRNA (50 nm for 48 h), incubated with the indicated inhibitor for 24 h, and harvested for immunoprecipitation-Western blotting to determine the levels of acetylated and total ER-α as in Fig. 3. B,Assays were performed as described for A, using the PI3-kinase inhibitor wortmannin. C, Assays were performed as described above, except that after treatment with siRNA, the cells were transfected overnight with DN-Akt or empty pcDNA3 vector before immunoprecipitation (IP)-Western blotting. D, Assays were performed as described for C, except that the cells were transfected with wtAkt rather than DN-Akt. E, Western blot showing the expression of the DN-Akt and wtAkt proteins after vector transfections. The inhibitors tested (concentrations) were as follows:LY294002 (100 μm), PD98059 (30 μm), GF109302X (30 μm), SB202190 (10 μm), rapamycin (10 ng/ml), PP1 (10 μm), and wortmannin (100 nm).