L929 cells or wild-type, Bid-deficient, or TRAIL-R-deficient MEFs were either adsorbed with reovirus at an MOI of 100 PFU/cell or mock-infected in serum-free medium at 4uC for 1 h, followed by incubation in serum-containing medium at 37uC for various intervals. Whole cell lysates were prepared by washing cells in phosphate-buffered saline (PBS) followed by lysis using 16 RIPA buffer (50 mM Tris [pH 7.5], 50 mM NaCl, 1% TX-100, 1% DOC, 0.1% SDS, and 1 mM EDTA) containing a protease inhibitor cocktail (Roche). Following centrifugation at 15,0006g to remove debris, the lysates were resolved by electrophoresis in polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked for at least 1 h in blocking buffer (PBS containing 5% milk or 2.5% BSA) and incubated with antisera against Bid (1:1000), actin (1:2000), or procaspase-9 (1:1000) either at room temperature for 1 h or 4uC overnight. Membranes were washed three times for 10 min each with washing buffer (PBS containing 0.1% Tween-20) and incubated with1:2000 dilution of horseradish peroxidase (HRP)-conjugated or Alexa Fluor-conjugated goat anti-rabbit Ig (for procaspase-9) or donkey anti-goat Ig (for Bid and actin) in blocking buffer. Following three washes, membranes were incubated for 1 min with chemiluminescent peroxidase substrate (Amersham Biosciences) and either exposed to film (for HRP-conjugated secondary antibodies) or scanned using an Odyssey Infrared Imager (LiCor).