Cell lysate was boiled in a sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol), and protein concentration was determined using a Bradford protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as the standard [47 (link)]. Following protein transfer, the membrane was blocked with 5% skim milk in PBS Tween- (PBST-) 20 for 2 h at room temperature and then incubated overnight with antibodies at 4°C (the primary antibodies include Bcl2, 1 : 1000, Cell Signaling Technology, #3498; Bax, 1 : 1000, Cell Signaling Technology, #2772; caspase-9, 1 : 1000, Cell Signaling Technology, #9504; c-IAP, 1 : 1000, Cell Signaling Technology, #4952). The membranes were then washed with PBST containing 0.1% Tween. After three washes in PBST, each blot was incubated with peroxidase-conjugated secondary antibody for 1 h at 37°C. Labeled proteins were visualized using the Odyssey infrared scanner (LI-COR, Lincoln, NB, USA) [48 (link)]. Signals were densitometrically assessed and normalized to the β-actin signals, and an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ, USA) was used to visualize the antibody-specific proteins in accordance with the manufacturer's recommended protocol [49 (link)].
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