(A–D) Wt and Bak−/−Bax−/− MEFs were treated with β-ME, DTT, Tg, or Tun for 18 h. (E) Wt, Bak−/−, and Bax−/− MEFs were treated with β-ME (15 mM), DTT (5 mM), Tg (1.5 μM), or Tun (2.5 μg/ml) for 18 h. (F) Lysates from ER stress treated Wt, Bak−/−, and Bax−/− MEFs in E were analyzed by western blot. (G) CHAPS lysates from ER stress treated Wt MEFs (highest doses at 18 h) were subjected to 6A7 IP and western blot. Total cell lysates (5%) were analyzed as a loading control. (H) HM fractions isolated from ER stress treated Wt MEFs (highest doses at 18 h) were subjected to trypsinization, and analyzed by western blot. Total cell lysates (5%) were analyzed as a loading control for BAX, VDAC is a pre-trypsinization mitochondrial loading control. (I) Wt and Bid−/−Bim−/− MEFs were treated with Tg for 18 h. (J) HM fractions isolated from ER stress treated Wt MEFs (highest doses at 18 h) were analyzed by western blot. (K) HM fractions from ER stress treated Wt MEFs (highest doses at 18 h) were incubated with ABT-737 (1 μM) for 30 min at 37°C, centrifuged, and the supernatants were analyzed by western blot. CHAPS (0.25%) lysed mitochondria indicates total cyto c within each lane. *Indicates a non-specific band. (L) Same as J, but probed for PUMA and SMAC. (M) Wt and Puma−/− MEFs were treated with Tg for 18 h. (N) Puma−/− MEFs were pre-treated with ABT-737 (1 μM) for 1 h, then β-ME for 18 h; or β-ME for 18 h, then ABT-737 for an additional 6 h. (O) A summary schematic of BCL-2 family interactions required for apoptosis to proceed. All data are representative of at least triplicate experiments, and reported as ± S.D., as required. See also .