Reovirus Activates a Caspase-Independent Cell Death Pathway

Berger, Angela K.; Danthi, Pranav

Immunoblot assay. The cell lysates or extracts 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% bovine serum albumin [BSA]) and incubated with antisera against Bid (1:1,000), RelA (1:1,000), IB␣ (1:500), NS (1:750), cleaved caspase-3 (1:500), PARP (1:1,000), HMGB1 (1:500), tubulin (1:500), or PSTAIR (1:10,000) at 4°C overnight. Membranes were washed three times for 5 min each with washing buffer (Tris-buffered saline [TBS] containing 0.1% Tween-20) and incubated with a 1:20,000 dilution of Alexa Fluor-conjugated goat anti-rabbit Ig (for RelA, IB␣, caspase-3, HMGB1, and PARP), goat anti-mouse Ig (for PSTAIR and tubulin), donkey anti-goat Ig (for Bid), or IRDye-conjugated anti-guinea pig IgG (for NS) in blocking buffer. Following three washes, membranes were scanned and quantified using an Odyssey infrared Imager (LI-COR).