Apoptosis Protein Expression Analysis

For detection of the expression of apoptosis relevant proteins, cells were grown to 70% confluence and lysed in RIPA buffer. The lysates (30 μg total protein per lane) were separated by SDS–PAGE, blotted onto PVDF-membrane and incubated with the appropriate primary antibodies followed by incubation with the HRP-conjugated secondary antibody (Amersham Pharmacia Biotech, Freiburg, Germany). Antigen visualization was performed by enhanced chemiluminescence (ECL-kit, Amersham Pharmacia Biotech). Primary antibodies used and their suppliers were as follows: anti-caspase-8 and anti-caspase-9 (StressGen Biotechnology, Canada), anti-caspase-3, anti-caspase-2, anti-caspase-7, anti-FADD, anti-Bcl-x_L, anti-Apaf-1, anti-BAD (Transduction Laboratories/BD, Heidelberg, Germany), anti-Bfl-1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bid, anti-c-FLIP, anti-XIAP, anti-c-IAP2 and anti-Survivin (R&D Systems, Wiesbaden, Germany), anti-Smac/DIABLO (Biocarta, Hamburg, Germany) and anti-Bax (PharMingen, Hamburg, Germany).