Cells were harvested and lysed using SDS lysis buffer [62.5 nM tris (pH 6.8), 1% SDS, 0.005% bromophenol blue, 4% glycerol, and 1% (v/v) ß-mercaptoethanol]. Samples were then vortexed and incubated at 95° for 5 min. Lysates were normalized to cell number and subjected to SDS polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and blocked with buffer consisting of TBS (tris-buffered saline)–T with 5% fat-free milk powder. The membranes were then incubated with primary antibodies against caspase-3, -7, -6, -8, or -9 (catalog nos. 9662S, 9492S, 9762S, 9746S, or 9502S, respectively; Cell Signaling), cIAP (catalog no. 3130; Cell Signaling), Bid (catalog no. 2002S; Cell Signaling), or tubulin (catalog no. 081 M4861, 1:1000; Sigma-Aldrich). After washing the membranes with TBS-T at room temperature, we then added the secondary antibodies (1:5000) EasyBlot anti-rabbit or anti-mouse (catalog nos. GTX221666-01 and GTX221667-01, respectively; GeneTex). The reactive bands were visualized by chemiluminescence and captured using Imager Lab.

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