Effect of Apaf-1 protein levels on cytochrome

c–mediated cleavage of exogenous procaspase-9.(A) Cytochrome c–induced procaspase-9 cleavage in the presence of S-100 from different cell lines. In vitro translated [³⁵S] procaspase-9 cleavage was initiated by the addition of cytochrome c and dATP in the presence of S-100 from cell lines. (B) Cytochrome c–mediated procaspase-9 cleavage in the absence of Apaf-1. The S-100 in which Apaf-1 was depleted has been indicated as ID. Failure to cleave procaspase-9 by cytochromec is shown by the absence of the cleavage bands (P35 and P37) compared with untreated S-100. (C) Purified Apaf-1 protein enhanced cytochrome c–induced procaspase-9 activation in K562 and CEM cell lines. 1 μL Apaf-1 protein was added to the S-100 (50 μg protein) lysates from K562 and CEM cells. Cytochromec–mediated procaspase-9 cleavage was allowed to proceed for 1 hour. Enhanced procaspase-9 cleavage is shown as disappearance of the procaspase-9 and P37 bands.