FIGURE 5.
BH3 domain mutations destabilize pro-survival proteins. A, pro-survival proteins with “pro-apoptotic” BH3 domains do not kill Bax−/−/Bak−/− MEFs, even when co-expressed with BimS. Colonies were scored, and numbers were expressed as a percentage of the number observed in cells transduced with the pMIG vector only. Error bars, S.D. of n = 3 assays. B, mutation of the h1 + 1, h3 or both residues in pro-survival proteins significantly impacts steady-state levels of FLAG-tagged pro-survival proteins in Bax−/−/Bak−/−MEFs. C, the reduced levels of pro-survival BH3 mutants are due to their shorter half-life as a result of them being more rapidly degraded by the proteasome following cycloheximide (CHX) treatment. In B and C, Western blots (WB) of equivalent cell lysates were probed with anti-FLAG antibody and then reprobed with anti-β-actin antibody to control for sample loading. The asterisk in C indicates a nonspecific band that becomes apparent due to the longer exposure of this blot.