Immunoblotting for procaspase processing. Whole cell extracts were prepared from 1 × 10⁶ neutrophils (PMNs) stimulated with TNF- plus cycloheximide (TNF/CHX) for the indicated time periods. (A) Pro–caspase-7 is not detectable in neutrophils. Whole cell extracts from 5 × 10⁵ Jurkat cells treated with (lane 5) or without (lane 6) anti-Fas antibody were also analyzed. (B) Processing of pro–caspase-8 (upper panel) and pro–caspase-3 (lower panel) in TNF-–treated neutrophils. Lane 6, neutrophils were preincubated with zVAD-fmk before exposure to TNF- plus cycloheximide. The same blot was sequentially probed with anti–caspase-8 (upper panel) and anti–caspase-3 (lower panel) monoclonal antibodies.