Figure 3.

Inhibition of Cas proteolysis in vivo by ZAVD-fmk and DEVD-cmk. (A) Rat-1 cells were pretreated for 3 h with the indicated concentrations of ZVAD-fmk or DEVD-cmk and then exposed to 40 μM etoposide for an additional 12 h. Cas proteolysis was analyzed by immunoblot analysis using Cas mAb as a probe. The same membrane was then stripped and reprobed with FAK mAb. Both antagonists effectively inhibited Cas and FAK cleavage. Molecular mass standards are indicated by the small arrowheads on the left. Original proteins (arrows) and cleavage fragments (large arrowheads) are shown on the right. (B) Apoptotic cell lysates (30 μg) were obtained from etoposide-treated cells for 36 h and incubated with in vitro–translated Cas in the presence of either ZVAD-fmk or DEVD-cmk. Both caspase inhibitors completely inhibited the cleavage of in vitro–translated Cas even at 50 nM.