FIGURE 5.
b-VAD-fmk fails to affinity-label any initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. A and B, Apaf-1-deficient cells (2.5 × 106/ml) were left untreated or incubated in the presence or absence of 120 μm b-VAD-fmk for 3 h at 37 °C and subsequently incubated at a density of 106/ml in the presence of 100 ng/ml agonistic anti-Fas antibody for 6 h (A) or heated for 1 h at 44 °C followed by a 6-h incubation at 37 °C (B). Cells were then harvested, lysed, and incubated with streptavidin-Sepharose beads, after which bound proteins were eluted and subjected to Western blot analysis. Casp, caspase; SAV, streptavidin.