FIGURE 3.

eIF4B Asp⁵⁶³ is a true caspase-2 cleavage site. Processing of wild type eIF4B and its mutants upon co-expression with caspases is shown. HEK 293T cells were transiently transfected with the indicated variants of FLAG-tagged eIF4B and caspase expression vectors. Cleavage of eIF4B was assessed by Western blotting with anti-FLAG antibody. Expression of caspases was confirmed using their respective antibodies. GFP served as a loading control. Intact poly(ADP-ribose) polymerase (PARP) indicates that cells had not reached late apoptotic phase. The inability of caspase-2 to cleave the eIF4B D563A mutant (EIF4B D563A), in contrast to wild type eIF4B (EIF4B WT), indicates that Asp⁵⁶³ is a true caspase-2 cleavage site. Results are representative of at least three independent experiments.