Mouse anti bcl 2 c 2

HeLa cell cultures were exposed to either solvent (0.1 % DMSO), or TAX, VBL and each of the ATIs at the indicated concentrations. After 24 hours protein extracts were separated through SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, blocked and incubated with the following primary antibodies, all screened using TAX-treated extracts: mouse anti-MCL-1 (22) (sc-12756, 1:200 over night), mouse anti-BCL-2 (C-2) (sc-7382, 1:500 over night), mouse anti-BAX (B-9) (sc-7480, 1:500 over night) and goat anti-actin (I-19) (sc-1616, 1:400), all from Santa Cruz Biotechnology; rabbit anti-cleaved Caspase-3 (Asp175) (#9661, 1:500 over night) and rabbit anti-Caspase-9 (#9502, 1:1000 over night) both from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse 1:10000 for anti-MCL-1 and anti-BAX or 1:2000 for anti-BCL-2, goat anti-rabbit 1:2000 and donkey anti-goat 1:5000 from Santa Cruz Biotechology) were revealed with enhanced chemiluminescence (ECL) or ECL plus (GE Healthcare, Chalfort Saint Giles, UK).

Samples were resolved by SDS–PAGE (15%) and electroblotted onto a 0.45 μm PVDF membrane (GE Healthcare). Primary antibodies used in this study included rabbit anti-BAX N20 (Santa Cruz Biotechnology), mouse anti-BCL-2(C2) (Santa Cruz Biotechnology) and mouse anti-cytochrome c antibodies (clone 7H8.2C12, Millipore). Detection was achieved using sheep anti-mouse IgG HRP (GE Healthcare) and donkey anti-rabbit IgG HRP (GE Healthcare) secondary antibodies. Proteins were visualized by 4CN Plus Chromogenic Substrate (PerkinElmer).