Expression of small t enhances Bcl2 phosphorylation and suppresses Bcl2/p53 binding in association with prolonged cell survival. (A) The pCMV5/small t construct or vector-only control was transfected into H7 cells expressing WT Bcl2 using Lipofectamine 2000. Expression levels of small t antigen were analyzed by Western blot using a small t antibody. (B) H7 WT Bcl2 cells expressing small t antigen or vector-only control were metabolically labeled with ³²P-orthophosphoric acid for 120 minutes. Bcl2 was immunoprecipitated using an agarose-conjugated Bcl2 antibody. Phosphorylation of Bcl2 was determined by autoradiography (upper). Western blot analysis was performed to confirm and quantify Bcl2 protein (lower). (C) H7 WT Bcl2 cells expressing small t antigen or vector-only control cells 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. p53, Bcl2, and Bax were then analyzed by Western blotting. (D) H7 WT Bcl2 cells expressing small t antigen or vector control 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.