Bcl x

Cells were harvested in a RIPA buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 1.0% (v/v) Igepal CA-630 (NP-40), 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) sodium dodecyl sulphate (SDS) supplemented with protease and phosphatase inhibitor cocktails from Sigma-Aldrich. The samples were then diluted in 3× sample buffer [187.5 mM Tris-HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue]. For the detection of PARP, cells were harvested in whole cell lysis buffer containing 62.5 mM Tris (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 0.00125% (w/v) bromophenol blue and 50 mM DTT and the samples sonicated briefly. All extracts were denatured at 65°C for 10 min before separation of proteins on a polyacrylamide gel and transfer to PVDF membrane. The PVDF transfers were probed overnight at 4°C with primary antibodies and then incubated with horseradish peroxidase-conjugated anti-mouse or anti rabbit secondary antibodies (Promega, Madison, WI, USA). Protein expression was visualised by enhanced chemiluminescence. All primary antibodies were supplied by Cell Signaling Technology Inc. (Beverly, MA, USA) except for those generated against PARP, Bcl-2, pro-caspase-3, c-FLIP, β-tubulin and GAPDH which were obtained from Merck Biosciences and Bcl-x, and BubR1 from BD Biosciences (Bedford, MA, USA).