Anti bcl xl

Cells were harvested with accutase treatment when adequate, washed with PBS, lysed and heated at 95 °C for 5 min (Laemmli buffer), 70 °C for 10 min (Bolt sample buffer) or 85 °C for 2 min (Tricine sample buffer). Extracts were separated by SDS-PAGE either on 12.5% gels (EZ-run, Fisher Scientific, Schwerte, Germany), 4–12% Bolt gels (Life Technologies) or 16% Tricine gels (Life Technologies), and proteins were transferred onto nitrocellulose membranes (0.2 μm). Membranes were probed with anti-mouse A1 (rat monoclonal, kindly provided by Marco Herold, WEHI, Melbourne, Australia), anti-human A1 (rabbit polyclonal, kindly provided by Jannie Borst), anti-mouse Mcl-1 (Rockland, Limerick, PA, USA, #600-401-394), anti-human Mcl-1 (BD, Biosciences, Heidelberg, Germany, #559027), anti-mouse Bcl-2 (BD, clone 3F11), anti-Bcl-x_L (NEB, Frankfurt, Germany, clone 54H6) or anti-GAPDH (Millipore, Darmstadt, Germany, clone 6C5) or anti-β-actin (Sigma, clone AC-15) antibodies. Proteins were visualized using peroxidase-conjugated anti-rabbit (Sigma), anti-mouse (Dianova), anti-hamster (Dianova) or anti-rat (NEB) antibodies by enhanced chemoluminescence detection (ECL Prime, GE Healthcare, Dornstadt, Germany; SuperSignal West Femto Substrate, Pierce, Fisher Scientific, Schwerte, Germany).

Anti–Bcl-2 (Ab-1) antibody was purchased from Merck Biosciences (Darmstadt, Germany). Anti-Bcl-X_L, anti-Bcl-w, anti-Bid, anti-Bim (C34C5), anti-Bak, anti-caspase-3, anti-caspase-8 (1C12), anti-caspase-9 and HRP-conjugated secondary antibodies were obtained from New England Biolabs (Berlin, Germany). Anti-Mcl-1 (22) antibody was from BD Bioscience (Heidelberg, Germany), anti-Bax (2D2) was from Santa Cruz Biotechnology (Heidelberg, Germany) and anti-cytochrome c (clone 7H8.2C12) was purchased from BD Biosciences (Heidelberg, Germany). Anti-β-actin (AC-15) and anti-α-tubulin (DM1A) antibodies were obtained from Sigma-Aldrich (Munich, Germany). Bax activation was measured via flow cytometry by staining with an antibody specific for the conformation of activated Bax (Clone 3; BD Pharmingen, Heidelberg, Germany). PCR primers and siRNAs were purchased from Eurofins MWG Operon (Ebersberg, Germany).