Fig 2: AIF, ETFB, ECHS1 and CS protein patterns in blue-native gels. High correlation in the distribution patterns for proteins AIF, ETFB, ECHS1 and CS is showed according with their BNE mobility in heart, liver, brain and fC57 the cell line.

Fig 3: In situ proximity ligation assay of Cx43, ETFB and AIF proteins in freshly isolated cardiomyocytes from WT and Cx43KICx32 mice. (A) Cells treated with no primary antibody (negative control) or with only anti-Cx43 were used to determine the signal specificity, whereas Cx43-PanCadherin and Cx43-SDHA pairs of proteins were used as a positive control. In the lower group of confocal images, Cx43 interacts with AIF and ETFB, as well as AIF with ETFB in a Cx43-independent manner. (B) Assessment of the number of signals detected with composed images using 8–12 z-stacks for each experimental condition, of at least three independent cardiomyocyte isolation events for each genotype.

Fig 4: Western Blot analysis of protein co-immunoprecipitation and mitochondrial distribution of Cx43, ETFB and AIF in WT and Cx43KICx32 mice. (A) A total amount of 1 mg of heart mitochondrial protein extracts from WT mice was immunoprecipitated using anti-Cx43, anti-AIF and anti-ETFB antibodies. In left panels, lanes from 1 to 3 correspond to the input sample, the initial total (T), subsarcolemmal (SSM) or interfibrillar (IFM) protein extracts. Lanes from 4 to 12 correspond to the output sample—the immunoprecipitated fraction—analysed for the presence of each protein. A representative IgG control of Cx43-IP protocol is shown in right panels using the total protein extract as a sample. Of note, the Cx43 antibody used recognizes also the phosphorylated protein forms (C0 unmodified, C1 phosphorylated). Also, due to proteinase treatment to obtain the IFM fraction, AIF is partially truncated and noted as a double lower molecular weight band. (B) Protein content of Cx43, AIF and ETFB. Anti-SDHA was used as a mitochondrial loading control. (C) Sub-mitochondrial distribution of Cx43, AIF and ETFB proteins was examined after partial digestion of the outer mitochondrial membrane with 0.1 and 0.5 µg of proteinase K, as indicated. Na+/K+ ATPase reactivity was assessed to rule out any potential contamination from gap-junctional membranes, and SDHA and TOMM20 were used as an inner and outer mitochondrial membrane protein control, respectively. A representative image of at least three independent replicates is shown.