UCN-01 diminished the association of Bim_EL with Bcl-2 and Bcl-x_L, which is restored by coadministration of PD184352. (A) RPMI8226 cells were exposed to 150 nM UCN-01 plus or minus 5 μM PD184352 for 24 hours, after which cells were lysed in CHAPS buffer and immunoprecipitation (IP) performed using either anti–Bcl-2 or anti-Bim antibody as described in “Materials and methods.” Immunopreciptates were then subjected to Western blot analysis (WB) using anti-Bim or anti–Bcl-2 antibody, respectively. (B) RPMI8226 cells (top panels) were treated as described for panel A, and U266 cells (bottom panels) were exposed to 100 nM UCN-01 ± 5 μM PD184352 for 40 hours, after which the association between Bcl-x_L and Bim was evaluated by IP/WB analysis using anti-Bcl-x_L (for IP) and anti-Bim (for WB), respectively. (C) RPMI8226 and U266 cells were treated as described for panels A and B, after which IP/WB was performed to examine the conformational change of Bax and Bak using anti-Bax 6A7 or anti-Bak Ab-1 (for IP), and anti-Bax or anti-Bak (for WB), respectively. For IP/WB analysis in panels A-C, 200 μg protein per condition was employed for IP. IgG levels are shown to ensure equal loading of IP antibodies and transfer. Phosphorylated forms of Bim_EL are manifested by slowly migrating species (▼). The results of representative experiments are shown; 2 additional studies yielded equivalent results. (D) Alternatively, cells were lysed in digitonin buffer, and subcellular fractions were prepared as described in “Materials and methods,” after which Western blot analysis was performed to monitor localization of Bax in either cytosol and organellar membrane (pellet) fractions. Each lane was loaded with 20 μg protein; blots were stripped and reprobed with antiactin antibody to ensure equal loading and transfer. Two additional studies yielded equivalent results.