Irdye 680 or 800 labeled secondary antibodies

Samples for western blot were lysed in Laemmli lysis buffer (0.12 M Tris HCL [pH 8], 4% SDS, 20% glycerol, 0.05 μg/μL bromophenol blue, and 50 mM dithiothreitol) and boiled for 5 min. Protein contents were determined by the Bio-Rad protein assay (Bio-Rad Laboratories), and equal amounts of total lysate were analyzed by 12% SDS–polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-P and incubated with blocking buffer (Tris buffered saline/Tween-20 [TBS-T]) containing 2% low-fat milk for 1 hour before incubating with an antibody against Mcl-1 (BD Pharmingen), Bcl-XL (Transduction Laboratories), Bim (Stressgen Bioreagents), Bcl-2 (Enzo Lifesciences), p100/p52 (Cell Signaling), or β-actin (Santa Cruz Biotechnology) overnight at 4 °C in TBS-T. Blots were subsequently incubated with IRDye 680 or 800 labeled secondary antibodies (Li-Cor) for 1 hour. Odyssey Imager (Li-Cor) was used as a detection method according to the manufacturers’ protocols. For RT-PCR, total RNA was extracted using the TRIzol isolation method (Invitrogen). cDNA was generated using the SuperScript II cDNA synthesis kit (Invitrogen). Transcripts were amplified by PCR using an ABI 7500 RT-PCR system. We used Hprt as a housekeeping gene for normalization.