Fas cd95

Cells were harvested and lysed in cell lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5 mM sodium orthovanadate, 1X protease inhibitor cocktail (BD Bioscience, San Jose, USA)). Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane (Amersham biosciences, Waltham, Massachusetts, USA). The membrane was blocked with 5% skim milk (BioShop, Burlington, Canada) for 1 h at room temperature (RT), then probed with the appropriate primary antibodies. Antibodies against BID, BAX, Bcl-2, Bcl-XL, and FADD were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Caspase-3, Caspase-8, PARP, horseradish peroxidase (HRP) conjugated anti-rabbit, anti-mouse, and anti-rat antibodies were purchased from Cell Signaling Technology (Beverly, USA). Antibody against α-tubulin was purchased from Sigma-Aldrich, and Fas/CD95 was purchased from Proteintech, and Flag was purchased from Stratagene. The secondary antibodies were diluted at a 1:2000 ratio in 5% skim milk and incubated for 3 h at RT. Chemiluminescence signals were detected by a chemiluminescent system (Intron).