XIAP knockdown restores caspase activity. (A) Cell lysates of L1309 B cells, Jurkat T cells, and HL cells L428, L428-scr-shRNA, L428-XIAP-shRNA, L540, L540-scr-shRNA, and L540-XIAP-shRNA were prepared, and equal amounts of protein were examined for XIAP, Smac, and caspase-3 expression using respective specific antibodies. Reprobing for actin ensured equal loading of cell extracts. (B) Cytosolic extracts of L1309 B cells, Jurkat T cells, and HL cells L428, L428-scr-shRNA, and L428-XIAP-shRNA were prepared, and equal amounts of protein were incubated with grzB (20 ng) for 1 hour at 30°C. Cytosolic extracts were resolved by SDS-PAGE and subjected to Western blot analysis for caspase-3 by polyclonal rabbit anti-caspase-3 antibody. Reprobing for actin ensured equal loading. (C) Relative caspase-3 activity was analyzed in the cytosolic extracts after grzB treatment using DEVD-AFC as substrate. Data are mean ± SD values from 3 individual experiments performed in triplicate.