FIG. 1.

Leukemogenic tyrosine kinases inhibit recruitment of caspase-9 to Apaf-1. (A) Cell lysates were prepared from control Ba/F3 cells or Ba/F3 cells expressing Bcr-Abl, FLT3/D835Y, or Tel-PDGFRβ and incubated with 0 or 2.5 ng/μl cytochrome c (Cyt c) and 1 mM dATP. Caspase-3 activity was assayed by measuring cleavage of DEVD-pNA. (B) Lysates were incubated with 1 mM dATP and various concentrations of cytochrome c, and immunoblotting was performed for caspase-9 (C9) and caspase-3 (C3). Procaspases and cleaved caspases are indicated by arrows and arrowheads, respectively. (C) Total cell lysates from control Ba/F3 cells (C) or Ba/F3 cells expressing Bcr-Abl (B), FLT3/D835Y (F), or Tel-PDGFRβ (T) were immunoblotted with anti-Apaf-1, anti-caspase-9 (C9), and anti-caspase-3 (C3) antibodies. (D) Cell lysates were separated on a Superdex 200 column before and after incubation with 2.5 ng/μl cytochrome c and 1 mM dATP for 30 min. Immunoblotting was performed for Apaf-1 and caspase-9. Procaspase-9 and cleaved caspase-9 are indicated by arrows and arrowheads, respectively. (E) Ba/F3 cells were transfected with FLAG-tagged caspase-9 (C287S), and lysates were prepared. Immunoprecipitation (IP) was performed with or without addition of cytochrome c (2.5 ng/μl) and dATP (1 mM). Pellets were analyzed by immunoblotting with anti-Apaf-1 and anti-FLAG antibodies.