Fig. 5.

Bak interaction with mitofusins: changes during apoptotic treatment and effects of BH3 mutation. (A) HeLa cells were untreated or treated with 10 mM azide for 3 h. Whole lysates were collected with CHAPS buffer and subjected to immunoprecipitation using an anti-Bak antibody. The resultant immunoprecipitates were analyzed for Fis1, Drp1, Mfn1, Mfn2, and Bak by immunoblotting. (B) HeLa cells were transfected with Myc-Mfn1 or Myc-Mfn2. The cells were untreated or treated with azide to collect whole-cell lysates for immunoprecipitation using an anti-Myc antibody. The resultant immunoprecipitates were analyzed for Bak, Bax, Myc-Mfn1, or Myc-Mfn2 by immunoblotting. (C) Bax/Bak double-knockout MEFs were cotransfected with Mito-Red and Bak, mBak, or empty vector. Cells with mitochondrial fragmentation were examined and counted by fluorescence microscopy. (D and E) Bax/Bak double-knockout MEFs were cotransfected with Bak or mBak and Myc-Mfn1 or Myc-Mfn2. The cells were then untreated or treated with azide to collect whole-cell lysates for immunoprecipitation using an anti-Myc antibody. The resultant immunoprecipitates were analyzed for Bak, mBak, and Myc-Mfn1 or Myc-Mfn2 by immunoblotting. Results in A, B, D, and E are representatives of at least three separate experiments. Data in C are presented as means ± SD of three separate experiments. ∗, significantly different from the empty vector transfection group.