ABT-737 reaches its target, BCL-2, in sensitive and resistant cells, and increased MCL-1 and BFL-1 sequester BIM displaced from BCL-2 by ABT-737. (A) OCI-LY1 cells were treated with 1μM ABT-737 for 4 hours after pretreatment with 10μM ZVAD.fmk for 30 minutes as indicated. Cells were lysed with 1% CHAPS lysis buffer. Lysates were immunoprecipitated using BCL-2– and MCL-1–specific antibodies. The resulting immunoprecipitated and coimmunoprecipitated proteins were analyzed by immunoblot alongside whole-cell lysates. *IgG heavy chain. (B) OCI-Ly1 R10 cells were treated with DMSO or 1μM ABT-737 (+) for 4 hours. Cells were lysed with 1% CHAPS lysis buffer. A total of 100 μg of lysate were immunoprecipitated with the BCL-2 (6C8) antibody. The resulting immunoprecipitated and coimmunoprecipitated proteins were analyzed by immunoblot alongside whole-cell lysates. These results are representative of 3 independent experiments. (C) OCI-Ly1 R10 cells were treated and lysed as in panel B. A total of 100 μg of lysate were immunoprecipitated with an MCL-1–specific antibody. The resulting immunoprecipitated and coimmunoprecipitated proteins were analyzed by immunoblot alongside whole-cell lysates. These results are representative of 3 independent experiments. (D) SU-DHL-4 cells were treated with DMSO or 1μM ABT-737 (+) for 12 hours after pretreatment with 10μM ZVAD.fmk for 1 hour. SU-DHL-4 R2 cells were treated with DMSO or 1μM ABT-737 (+) for 12 hours. Cells were lysed with 1% CHAPS lysis buffer. Lysates were immunoprecipitated using a BCL-2–specific antibody (6C8). The resulting immunoprecipitated and coimmunoprecipitated proteins were analyzed by immunoblot. (E) SU-DHL-4 and SU-DHL-4 R2 cells were treated and lysed as in panel D. Lysates were immunoprecipitated using a BIM-specific antibody. The resulting immunoprecipitated and coimmunoprecipitated proteins were analyzed by immunoblot. (F) Immunoblot analysis of the whole-cell lysates used for immunoprecipitation in panels D and E.