Anti fadd

Cells were harvested by centrifugation at 1200 r.p.m. for 5 min at 4 °C, washed with PBS and suspended in RIPA buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP40, 0.1% SDS and 0.5% sodium deoxycholate) containing protease inhibitor cocktail (Roche Lifesciences, Indianapolis, IN, USA). Samples were centrifuged at 14 000 r.p.m. for 15 min at 4 °C. The cell lysates were then separated by 10–15% SDS-PAGE and transferred to polymer of vinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA) and blocked with 5% non-fat milk in PBST (PBS with 0.5% Tween-20). Primary antibodies used include anti-p53, anti-caspase-3, anti-caspase-8, anti-Bax, anti-caspase-9, anti-XIAP, Rabbit polyclonal anti-PARP, anti-DR5 (all from Cell Signalling Technology, Inc., Danvers, MA, USA), anti-Mcl-1, anti-Bcl-2, anti-β-actin, anti-GAPDH, anti-ANT, anti-AIF, anti-Bcl-xL (all from Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA), anti-cIAP2, anti-FADD, anti-iNOS (all from BD Biosciences, San Jose, CA, USA), anti-DR4 (Upstate, EMD Millipore Corporation, Billerica, MA, USA), anti-caveolin, anti-flotillin (Abcam, Cambridge, UK), anti-cFLIP (Alexis, Enzo Life Sciences). After incubation with HRP-conjugated secondary antibody (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL, USA), protein expression was detected by a chemiluminescence reaction (EMD Millipore).