Figure 1.

Anti-connexin-43 antibody immunoreactivity in cardiac mitochondria. A. The anti-Cx43-immunoreactivity (red) of some low density cultured rat neonatal ventricular myocytes was peri-nuclear and punctate visible throughout the cell reminiscent of the MitoTracker green staining (green). Calibration bar = 20 μm. B. Crude homogenate of adult rat heart ventricles was separated by two rounds of centrifugation as described in the Methods. P1=first pellet, S1=first solute, P2=second pellet and equal protein amount (30 μg) was loaded in each lane. Br=total brain lysate served as a control and the immunoreactivity to the following proteins served as markers for cellular compartments: VDAC (mitochondrial marker), GAPDH (cytosolic marker), N-Cad (plasma membrane marker). C. The P2 pellet from the centrifugation was further fractionated by iodixanol gradient and equal volume from the 13 collected fractions was loaded and proteins analyzed by immunoblotting. The far right lane of the crude homogenate served as a positive control for the respective antibody immunoreactivity. D. The mitochondria present in the P2 pellet was separated into the sub-mitochondrial fractions as described in Methods. Equal protein amount (15 μg) from the respective fractions was loaded and proteins analyzed by immunoblotting. ANT (inner mitochondrial membrane marker), VDAC (outer mitochondrial membrane marker), IMM (inner mitochondrial membrane), CS (contact site), OMM (outer mitochondrial membrane), Br (brain). Representative of 3 Western blots from two separate mitochondrial membrane fractionation preparations. E. Mitochondrial lysate was subjected to dephosphorylation by alkaline phosphatase and probed with antibody recognizing all Cx43 regardless of the phosphorylation status of the protein (Pan-Cx Ab) or with non-phosphorylated-species specific antibody (P0-Cx Ab). Arrows point to the putative phosphorylated Cx43 species.