Overexpression of HA-tagged PP2A/C results in dephosphorylation of Bcl2, increased p53/Bcl2 binding, and apoptotic cell death. (A) H7 WT Bcl2 cells were metabolically labeled with 32P-orthophosphoric acid and treated with IL-3 for 15 minutes. Phosphorylated Bcl2 was immunoprecipitated and incubated with purified PP2A (10 ng) at 30°C for 10 minutes. Phosphorylation of Bcl2 was determined by autoradiography. (B) The HA-tagged PP2A/C–pCDNA3 construct or vector control was transfected into H7 WT Bcl2 cells using Lipofectamine 2000. Expression levels of exogenous HA-PP2A/C were analyzed by Western blot using an anti-HA antibody. (C) H7 WT Bcl2 cells overexpressing HA-PP2A/C or vector control were metabolically labeled with 32P-orthophosphoric acid and treated with IL-3 for 15 minutes. Phosphorylation of Bcl2 was analyzed by autoradiography. (D) H7 WT Bcl2 cells overexpressing HA-PP2A/C or vector control 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. (E) H7 WT Bcl2 cells overexpressing HA-PP2A/C or vector control were treated with cisplatin (30 μM) for 24 hours. Cell viability was determined by analyzing annexin V binding on FACS. Data represent the mean plus or minus SD of 3 separate determinations.
