Cell medium was removed, and cells were washed twice with cold Hanks’ balanced salt solution and lysed with RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN). Lysates were transferred to 1.5-mL tubes (Eppendorf, Fremont, CA) and cleared by centrifugation. Total protein in the supernatants was measured by Bradford assay (Bio-Rad) with bovine serum albumin (BSA) used to generate the curve, according to the manufacturer’s instructions. Protein (30 μg) was electrophoresed on a 12.5% SDS-polyacrylamide gel overlaid with a 3.6% polyacrylamide stacking gel. The proteins were transferred to nitrocellulose membrane (Bio-Rad) with a mini transblot apparatus (Bio-Rad), according to the manufacturer’s directions. Recombinant human Bcl-2-related protein, A1 (R&D Systems, Inc.), and human whole-cell lysate from HL-60 acute promelocytic leukemia cells (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used as positive controls for A1 and Bcl-2 Western blot analysis, respectively. Transfers were performed overnight at room temperature (RT). Nonspecific binding sites were blocked by immersing the membrane in 5% fat-free milk powder (SACO Foods Inc., Middleton, WI) for 30 minutes at RT. The blocking step was repeated, and then membranes were washed three times (5 minutes per wash) in Tris-buffered saline (73 mM Tris, 40 mM NaCl [pH 7.45]) containing 0.1% Tween-20 (TBST). The membranes were incubated overnight at 4°C with the following antibodies (Santa Cruz Biotechnology, Inc.) diluted in 5% milk: rabbit polyclonal antibody directed against c-IAP1 (1:2000), c-IAP2 (1:2000), TRAF-1 (1:2000), TRAF-2 (1:1500), Bcl-x
L (1:800), Bcl-2 (1:800), survivin (1:800), A1 (1:500), and mouse monoclonal antibody against c-FLIP NF6 (1:500 in 5% milk; Alexis, San Diego, CA). The blots were then washed three times (20 minutes per wash) in TBST and incubated with anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (1:5000 in 5% milk; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 4°C for 60 minutes. Immunoreactive bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham, Piscataway, NJ). Data shown in
Figures 2 ,
Figures 3 ,
Figures 4 ,
Figures 5 ,
Figures 6 to
7are from a representative experiment. In all the figures with histograms of densitometry data, optical density (OD) refers to the integrated density. Experiments in duplicate were separately repeated twice with similar results.