Apoptotic Signaling Pathway Analysis

All cell culture and transfection reagents were from Invitrogen; and standard reagents were from Sigma or Fisher Scientific. Drugs were from: ABT-737 (Abbott Pharmaceuticals), ABT-263/PLX-4032/GSK-110212 (Selleck), zVAD-fmk (Calbiochem), Hoechst 33342 (Anaspec), and staurosporine/cisplatin/dacarbazine/vinblastine (Sigma). Antibodies (clone): anti-actin (C4), anti-A1 (FL-175), anti-BCL-2 (100), anti-CD31 (BD Pharmingen #550274) anti-MCL-1 (Rockland), anti-BAD (C7), anti-BIM (22 (link)–40), anti-PUMA (CT; Sigma), anti-SMAC (H177), anti-BID (C20), anti-cytochrome c (7H8.2C12), anti-BAK (G23), anti-BAX (6A7 for IP; N20 for western blot), anti-BCL-xL (H5 for IP; S18 for western blot), anti-GAPDH (9B3), anti-HSP60 (B-9), anti-p44/p42 MAPK ERK1/2 (137F5), and anti-phospho p44/42 MAPK ERK1/2 (197G2). Caspase-8 cleaved mouse BID (C8-BID) was from R&D Systems. Full-length BAX was made as described (24 (link)). Human BCL-xLΔC, MCL-1ΔC, and PUMAβ were made as described (17 (link)). The human BIM BH3 domain peptide (> 98% purity, Abgent) was resuspended in anhydrous DMSO in a N₂ environment, stored at −80°C, and thawed only once. All lipids for the LUVs studies were purchased from Avanti Polar Lipids. Statistical significance was evaluated by two tailed Student t test for p < 0.05.