Figure 3. Abrogation of iASPP cleavage in CEM cell-free extracts by caspase-3 depletion A.

CEM cell free extracts were treated with a pan-caspase inhibitor (Z-VAD-FMK) or specific caspase-3 inhibitor (Z-DEVD-FMK) 1 hour before triggering caspase cascade by adding cytochrome c and dATP. The cleavage of iASPP was detected by anti-v5 antibody. B. Caspase cascades were triggered at 37°C by adding Cyt C and dATP in the control, IgG depleted or caspase-3 depleted CEM cell-free extracts containing same amount of in vitro translated v5 tagged iASPP. Mixtures were separated by 8% SDS-PAGE followed by immunoblotting with the anti-v5 antibody. Immunodepletion efficiency was confirmed by western blotting with anti-caspase-3 antibody. The arrowheads indicate iASPP and cleaved iASPP fragments.