Fig. 3. Recruitment of caspase-3 to the Apaf-1 apoptosome requires catalytically active processed or unprocessed caspase-9. Caspase-9-depleted lysates were reconstituted with (A) fully processed wild-type p35 caspase-9, (B) non-cleavable D315/330A caspase-9 or (C) catalytically inactive C287A caspase-9. Following dATP activation for 1 h at 37°C, the reconstituted lysates were fractionated and immunoblotted for caspases-9 and -3, as described in Materials and methods. The arrows represent either the unprocessed or processed form(s) of the caspases; a non-specific band (*) was detected by the anti-caspase-3 antibody.