Fig. 2. Detection of caspase cleavage in whole cells and cell-free extracts. (A) 50-100 µg of whole lysates of U937 and ARPE-19 cells were analyzed by western blotting using antibodies against both pro- and cleaved forms of caspase-9 (top), -3 (middle) and PARP (bottom) in control cells and 4 hours after exposure to 50 µM menadione. (B) Cytosolic extracts of 5x10⁴ cells were prepared as described in Materials and Methods, incubated with 10 µM of cytochrome c and 1 mM ATP for 37°C for 2 hours, separated on a reducing gel and analyzed by western blotting using antibodies against caspase-9 (top) and -3 (bottom). (C) ARPE-19 cells were preincubated with Z-VAD.fmk (100 µM) for 1 hour and exposed to various concentrations of menadione (1 µM to 100 µM) for 2 hours. Cell viability was determined using the XTT assay as indicated in Fig.1A. (), menadione alone; (), menadione with Z-VAD.fmk.