Effect of caspase-8 on rECP-induced apoptosis. (A) Cells were treated with 20 μM caspase-8 inhibitor (Z-IETD) for 30 min before rECP treatment. After a 48-h incubation in the presence or absence of 20 μM rECP, total cell lysates were resolved by 10% SDS-PAGE. Relative cleaved PARP was assessed by western blotting using anti-PARP and anti-actin followed by quantitative analysis. (B) BEAS-2B cells were cultured for 48 h in the presence or absence of 20 μM rECP. Total cell lysates were resolved by 15% SDS-PAGE. Caspase-8 and PARP were analyzed by western blotting using anti-caspase-8 and anti-PARP. (C) rECP was incubated with proteinase K (PNK) (250 μg/ml) for 12 h followed by heating in boiling water for 10 min. Cells were treated in the presence or absence of rECP (20 μM), heated PNK (25 μg/ml), heated mixtures (20 μM rECP and 25 μg/ml PNK), and rECP H15A/K38I/H128A mutant (20 μM) for 48 h. All data represent the arithmetic mean ± SEM. ***P < 0.001.