FIGURE 4.

Cysteine 62 to serine substitution prevents cyt c release and caspase-3 processing associated with H₂O₂-induced apoptosis. A, analyses of cyt c release and Bax translocation. Cells were cultured on chamber slides in the presence or absence of 25 μm H₂O₂ for the times indicated, subjected to immunofluorescence staining with anti-cyt c antibody, and then analyzed by confocal microscopy. B, cells were cultured in the presence or absence of 25 μm H₂O₂ for 6 h and subjected to subcellular fractionation. The cytosolic fractions were immunoblotted (30 μg of protein/lane) with antibody specific for cyt c or caspase-3, which only detected the cleaved caspase 3. β-Actin was used as a protein loading control. Data are representative of at least three independent experiments.