DEVD-AFC and PARP cleavage activity assays using dephosphorylated and control extracts. (A) Measurements using a fluorogenic assay showed no differences between the ability of dephosphorylated (open symbols) and control extracts (closed symbols) to cleave the synthetic substrate DEVD-AFC. Inset, data replotted by the method of Lineweaver and Burke. (B) Dephosphorylated extracts exhibit enhanced ability to cleave PARP. (Top) PARP cleavage was examined by enhanced chemofluorescence. Lanes 1 and 2 correspond to PARP and extract incubated alone, respectively. (This extract contains the cleaved PARP fragment.) Lanes 3 and 4, cleavage of PARP by dephosphorylated extract. Lane 5, PARP was incubated in the presence of control extract following pretreatment with DEVD-fmk. (There is no significant decrease in intact PARP—the fragment is from the extract as in lane 2.) Lanes 6 and 7, cleavage of PARP by control extract. (Bottom) Quantitation of the PARP cleavage results (average of three independent experiments, with the standard deviations indicated). Downward black bars correspond to the relative decrease (relative to the starting level) in the amount of full-length PARP remaining after incubation for 20 minutes in phosphatase-treated and control extracts. Both the decrease in intact PARP and corresponding increase in the cleaved fragment are enhanced in phosphatase-treated extracts.