Caspase-2 processing correlates temporally with the induction of caspase-3-like activity. A, Lysates of PC12 cells (100 μg of protein) were prepared at successive times after serum deprivation in the absence or presence (N) of NGF, resolved by SDS–PAGE on a 12% polyacrylamide gel, and subjected to Western immunoblotting, using an antibody directed against the N terminus of caspase-2 (anti-N-Nedd; 1:250). The arrows indicate the N-terminal cleavage products. Positions of molecular weight standards are indicated at the margins of this and subsequent figures. B, The same lysates of PC12 cells as in A were prepared at successive times after serum deprivation, and 50 μg of protein from each lysate was incubated with the fluorogenic substrate DEVD-AFC (15 μm; ESP) (Stefanis et al., 1996). The release of AFC was quantified in a LS50B (Perkin-Elmer, Emeryville, CA) fluorimeter (excitation 400 nm, emission 505 nm). C, Particulate extracts (100 μg of protein) from the same set of cells as inA and B were prepared at successive times after serum deprivation, resolved by SDS–PAGE on a 10% polyacrylamide gel, and subjected to Western immunoblotting, using an antibody directed against PARP (C2–10; 1:10000; ESP). The arrowindicates the cleavage product of PARP.