Depletion of PP2A/C by RNAi enhances Bcl2 phosphorylation and suppresses Bcl2/p53 binding in association with increased cell survival. (A) PP2A/C siRNA or control siRNA was transfected into H7 WT Bcl2 cells using Lipofectamine-2000. Expression levels of endogenous PP2A/C were analyzed by Western blot using an anti-PP2A/C antibody. (B) H7 WT Bcl2 cells expressing PP2A/C siRNA or control siRNA were metabolically labeled with ³²P-orthophosphoric acid for 2 hours. Phosphorylation of Bcl2 was analyzed by autoradiography. (C) H7 WT Bcl2 cells expressing PP2A/C siRNA or control siRNA were treated with cisplatin (30 μM) for 24 hours followed by lysis in 1% CHAPS-containing buffer. Coimmunoprecipitation was performed using an agarose-conjugated Bcl2 antibody. The Bcl2-associated p53 and Bcl2 were analyzed by Western blotting. (D) H7 WT Bcl2 cells expressing PP2A/C siRNA or control siRNA were treated with cisplatin (30 μM) for 48 hours. Cell viability was determined by analyzing annexin V binding on FACS. Data represent the mean plus or minus SD of 3 separate determinations.