Bax 6a7

Samples grown over Nunc Lab-Tek chambers (Thermo Fisher Scientific, Waltham, MA, USA) were washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS and blocked with PBS 1% BSA-0.3% Triton for 60 min. Samples were then incubated overnight at 4 °C with primary antibody against BAX-6A7 (BD Bioscience, Franklin Lakes, NJ, USA), activated Caspase 3 (Cell Signaling, Danvers, MA, USA) or γH₂AX (Cell Signaling, Danvers, MA, USA) in PBS 1% BSA-0.3% Triton.
After three washes with PBS, samples were incubated with the corresponding FITC-conjugated (Activated Caspase-3 or γH₂AX) or TRITC-conjugated (BAX-6A7) secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. Samples were washed three times with PBS and cell nuclei were finally stained with DAPI (2 μg/mL). Images were captured using a ZEISS Axio Observer (ZEISS, Oberkochen, Germany) microscope and analyzed using the Carl Zeiss Microscopy GmbH’s ZEN 3.0 software.

Cells were lysed in CHAPS buffer (10 mM Hepes, pH7.5, 150 mM NaCl, 2% CHAPS) or RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1.0% NP-40) containing protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor cocktail I (Wako Pure Chemical, Osaka, Japan). Protein lysates (500 μg of protein) in CHAPS buffer were subjected to immunoprecipitation using an antibody to BAX 6A7 (BD Biosciences, San Jose, CA, USA), BAK Ab1 (Abcam), BIM (Cell Signaling Technology) or MCL1 (BD Pharmingen). The immunoprecipitates or whole cell lysates (10 μg of protein) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 7.5-15% gels and electroblotted onto nitrocellulose membranes (BIO-RAD, Hercules, CA, USA). Immunoblots were treated with primary and secondary antibodies and then analyzed using an ECL-Advance Western blotting kit (GE Healthcare, Little Chalfont, England). In several experiments, band intensities were quantified using a LAS4000 imaging system (GE Healthcare).