Figure 3.

The Mcl-1–specific Bim variant does not trigger Mcl-1 degradation. (A) Immunoblots of equivalent lysates prepared from MEFs after infection with parental retroviral vector or ones expressing wild-type Bim_S, Bim_S4E, Bim_S2A, Bim_SNoxaBH3, or Noxa itself were probed with antibodies to Mcl-1 (top), Bcl-2 (middle), or HSP70 (loading control; bottom). Levels of Mcl-1 were significantly lower if Noxa or Bim_SNoxaBH3 (Bim_S with its BH3 domain replaced with that from Noxa) is expressed. In contrast, the Mcl-1–specific Bim_S2A variant stabilizes Mcl-1 levels. The images were assembled and cropped from a single gel. (B) Bim_S2A counters Mcl-1 degradation triggered by Noxa. Equivalent lysates were prepared from Noxa- or Bim_S4E-overexpressing MEFs after reinfection with the inert Bim_S4E, Noxa, or Bim_S2A retroviruses, and the blots were probed for Mcl-1 or HSP70 (loading control). Note that Mcl-1 levels are significantly higher in cells expressing the Mcl-1–specific Bim_S2A. (C) ITC analysis of BimBH3 2A and NoxaBH3 binding to Mcl-1. BimBH3 2A binds to Mcl-1 tighter than NoxaBH3.