C5-treated mitochondrial supernatant does not induce caspase 3 activation in cell-free system of apoptosis.

Cytosolic extract (150 μg) from HL60 cells were incubated for 6 hours at 30°C in the presence of 1 mmol/L dATP with supernatants derived from purified mitochondria after treatment with buffer (Mt Sup), 150 μg C5 (C5 Sup.), or 100 μg C2 (C2 Sup.). Alternatively, cytosolic extracts (150 μg) were incubated with 0.1 μmol/L to 10 μmol/L Cyt C. Caspase 3 activation was assessed in the cytosols by the fluorometric assay (x increase) and by Western blot analysis of active caspase 3 (17 kd), as described in “Materials and methods.” Supernatants from C5- and C2-treated mitochondria were also analyzed for the presence of Cyt C by Western blotting using a monoclonal anti-Cyt C antibody.