C2-ceramide induces a conformational change of Bcl2 by exposing its BH3 domain in association with apoptosis. (A) H7 WT Bcl2 cells were treated with increasing concentrations of C2-ceramide for 24 hours. Coimmunoprecipitation was carried out using a Bcl2 BH3 or full-length Bcl2 antibody, respectively. Bcl2 or Bax was analyzed by Western blot using a Bcl2 or Bax antibody, respectively. (B) H7 WT Bcl2 cells were treated with C2-ceramide for 24 hours. Cells were then washed with 1× PBS, fixed and permeabilized with ice-cold methanol and acetone, and then blocked with 10% rabbit serum followed by staining with 4,6-diamino-2-phenylindole, a Bcl2 BH3 domain primary, and FITC-conjugated (green) secondary antibodies. Images were merged using Open-lab 3.1.5 software. (C) H7 WT Bcl2 cells were treated with increasing concentrations of C2-ceramide 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. (D) Purified Bcl2 protein was incubated with increasing concentrations of purified PP2A in 1% CHAPS lysis buffer at 4°C for 2 hours. Coimmunoprecipitation was carried out using a Bcl2 BH3 or full-length Bcl2 antibody, respectively. Bcl2 was analyzed by Western blotting.