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. 1998 Nov 10;95(23):13664–13669. doi: 10.1073/pnas.95.23.13664

Figure 2.

Figure 2

Oncogene-dependent caspase activation in the cell-free system requires caspase-9 and Cyt c. (A) Depletion of caspase-9. The 293-extract (Input) was depleted of caspase-9 by using protein A Sepharose bound to a caspase-9 antibody (α-casp9) or was mock-depleted by omitting the antibody. Caspase-9 remaining in extracts (S) and bound to the Sepharose (P) was assessed by immunoblotting by using the same antibody. The two additional bands are IgG. (B) Depletion of caspase-9 abolished caspase activation, which was restored by adding recombinant caspase-9. Data are typical of three experiments. (C) Depletion of Cyt c. The FT fraction (input), used in the reconstitution assay, was depleted of Cyt c by passing it over a phosphocellulose column. The resultant flow through (FT-P) had no Cyt c detectable by immunoblotting. (D) Cyt c is required for caspase activation. FT-P was mixed with ATP (1 mM) and APAF-1 (≈1 fmol) with or without Cyt c (≈20 pmol) as indicated. Results shown are typical of two experiments.