Mutation of threonine 125 does not prevent caspase 9 phosphorylation and cleavage inhibition. (a) CCL39-Raf-1:ER cells were transfected with the indicated construct (CD8 is used as a transfection control) and 48 hours later were deprived of serum in the presence or absence of 1 M 4-HT for 24 hours. The cell lysates were analyzed by immunoblotting for myc and phospho-MAPK1,3. (b) CCL39-Raf-1:ER cells stably expressing myc-tagged C287S (catalytically inactive) caspase 9 or C287S-T125A caspase 9 were incubated for 2 hours with 1 M 4-HT or 10 M U0126 in serum-free medium. Total cells were lysed in 2D sample buffer containing 6 M urea, resolved by two-dimensional gel electrophoresis, and analyzed by immunoblotting for myc.