Figure 5
From: The porin VDAC2 is the mitochondrial platform for Bax retrotranslocation
Bax and VDAC2 are present in common complexes on the OMM.
(A) BN-PAGE analysis of isolated mitochondria from wild-type (WT), Bax/Bak DKO, VDAC2 KO (2KO) or VDAC1/3 DKO (1/3DKO) MEFs solubilized in digitonin buffer by anti-Bax (left) or anti-VDAC (right) antibodies. The apparent molecular weight (MW) of the protein complexes is indicated. VDAC-staining of VDAC1/3 DKO cells (broken line box) is shown also with a long exposure (right). Complexes containing VDAC2 and Bax are indicated by arrows on the left. n = 3. (B) Short (left) and long (right) exposures of the Western blot analysis of antibody induced shifts in BN-PAGE migration using anti-VDAC and anti-Bax antibodies. Bax-containing protein complexes from wild-type (WT) MEFs and samples from Bax/Bak DKO MEFs were analyzed with or without prior incubation with anti-Bax 2D2 antibody (Sigma Aldrich). Molecular weight (MW) of the protein complexes is indicated on the right. Shifted protein complexes are indicated by white arrows. n = 3. (C) BirA fusion assay used to identify transient Bax-interacting proteins in human cells in the absence and the presence of overexpressed Bcl-xL. Western blot analysis of heavy membrane (mitochondria) and cytosolic fractions using indicated antibodies reveals BirA-Bax interactions with VDAC2 and Bcl-xL. myc-BirA-Bax is analyzed using anti-myc antibodies. n = 3. (D) BirA-Bax (blue) fusion expression in human cells leads to the labeling of VDAC2 (purple) and Bcl-xL (red) in close proximity to Bax with biotin (yellow asterisk) by BirA. Labelled proteins are present in the cytosolic fraction as they retrotranslocate after transient contact.
