Vdac Western blots. Western blots of VDAC isoforms in (1) WT CD1/J6/129svJ myocytes, (2) WT BL6 myocytes, and (3) VDAC1-deficient CD1/J6/129svJ myocytes were performed as described previously using a radioimmunoprecipitation assay buffer optimized for cardiac myocytes (Deisl et al., 2019). A nonselective VDAC1/2 polyclonal antibody, 10866-1-AP (Proteomtec, 1:3,000 dilution), and a selective VDAC3 antibody, 55260-1-AP (Proteintech, 1:1,000), were employed. Either β-actin (mouse monoclonal, SAB1305554, 1:200 dilution; Sigma-Aldrich) or α-actinin was blotted as loading control (mouse monoclonal antibody, EA-53, 1:200 dilution; Sigma-Aldrich). All wells were loaded with 70 μg of protein, primary antibodies were incubated overnight (4°C), and secondary antibodies (Amersham ECL HRP-conjugated) were incubated at 24°C for 2 h (1:10,000 dilution). To calculate the ratios of VDAC3 to α-actinin accurately, the relative abundances of α-actinin were first determined using a low enough exposure to avoid saturation (red) with subtraction of neighboring baseline (lower right blot). A higher exposure was used to determine the relative abundances of VDAC3 with equivalent baseline subtraction (lower left blot). Then, the density of bands showing no saturation was determined in the two images with subtraction of the background, and the average ratio of densities between the images was calculated. Expression values for α-actinin from the right blot were multiplied by that ratio, and the final abundancy ratios given were calculated from the image-adjusted α-actinin and VDAC3 values obtained in the lower left blot. Source data are available for this figure: SourceData FS5.