FIG. 7.

Bcr-Abl inhibits caspase 9 recruitment to the apoptosome. GST fusion proteins containing the prodomain of caspase 9 (A), the CARD of Apaf-1 (B), or Apaf-1 (1-543) (G) were incubated with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing lysates in the presence of 1 mM dATP. GST proteins were then rebound to glutathione beads (GSH), and the binding ability of Apaf-1 (A) or caspase 9 (B and G) was examined via immunoblot analysis. (C) GST-Apaf-1 (1-543), GST, or GST-Crk protein was incubated with [γ-³²P]ATP in either control GFP (lane C)- or Bcr-Abl (lane B)-expressing cell lysates, and radiolabeling was assessed via autoradiography. (D) Apaf-1 was immunoprecipitated from either control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates, and precipitates were immunoblotted (IB) for Apaf-1 or phosphotyrosine (P-Tyr). Anti-Abl immunoprecipitates (IP) were also immunoblotted for P-Tyr as a positive control for the phosphotyrosine Western analysis. (E and F) Control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates were incubated with cytochrome c-Sepharose and 1 mM dATP. The binding ability of Apaf-1 (E) and caspase 9 (F) was then examined by immunoblot analysis.