Fig. 2.

Bax D68R is functional but deregulated. (A) Like Bax, the D68R mutant is predominantly cytosolic but becomes membrane integrated after an apoptotic stimulus. Untreated or etoposide-treated (10 μM for 24 h) bax^−/−bak^−/− MEF stably expressing FLAG-tagged Bax or Bax D68R were separated into cytosolic (c), membrane-associated (a), and the carbonate-resistant membrane-integrated (i) fractions (see Materials and Methods). HSP70, cytosolic marker; VDAC1, integral mitochondrial outer membrane protein; GAPDH, loading control. *VDAC1 bands carried over from a previous blot. (B) Bax D68R retains full apoptotic function. The viability of reconstituted bax^−/−bak^−/− MEF (described in A) after etoposide treatment (0–10 μM) for 24 h was assessed by propidium iodide exclusion using flow cytometry. (C) Selectivity of BH3-only ligands for their prosurvival targets (see text). (D) Neutralization of Bcl-x_L, Bcl-w, or both activates Bax D68R but not WT Bax. Viability of reconstituted bax^−/−bak^−/− MEF was determined 24 h after infection with retroviruses expressing the indicated BH3-only proteins. Data in B and D represent means ± 1 SEM of 3 or more independent experiments.