Caspase-2 is processed by a noncaspase-3-like caspase. A, PC12 cells were deprived of serum in the presence or absence of various concentrations of DEVD-FMK or 100 μm CPT-cAMP. Six hours later, the cells were harvested for assessment of PARP cleavage (top panel), using immunoblotting with the C2–10 antibody (1:10000), and for caspase-2 cleavage (bottom panel), using immunoblotting with anti-N-Nedd (1:250). The arrowsdenote the cleavage products of PARP and caspase-2. Lysates of these cells were also used for measurement of DEVD-AFC cleavage activity, as described in Materials and Methods. The activities are reported relative to those of untreated controls. Replicate cultures from the same set of cells were used for assessment of survival (n = 3). B, PC12 cells were deprived of serum in the presence or absence of CPT-cAMP (100 μm) or the indicated concentrations of DEVD-FMK, zVAD-FMK, or BAF. For all three caspase inhibitors, a 4 hr pretreatment was also applied to the cells. Twelve hours after serum deprivation, lysates were harvested for the assessment of PARP processing, using immunoblotting with the C2–10 antibody (1:10000). The arrow denotes the cleavage product of PARP. C, PC12 cells were deprived of serum in the presence or absence of CPT-cAMP (100 μm) or DEVD-FMK (50 μm, with pretreatment for 4 hr). Twelve hours later, lysates were harvested for assessment of caspase-2 processing, using immunoblotting with anti-N-Nedd (1:200). The arrowdenotes the cleavage product of 37 kDa. D, PC12 cells were deprived of serum in the presence or absence of 100 μm zVAD-FMK, BAF, or CPT-cAMP. Six hours later, the cells were harvested for assessment of caspase-2 cleavage, using immunoblotting with anti-N-Nedd. The arrow denotes the N-terminal cleavage product.