(A) Schematic of predicted OCIAD1 and OCIAD2 topologies. (B) Representative images of fixed U2OS cells stained with Mitotracker (magenta) and immunolabeled for OCIAD2 (green). The bottom panel is a magnification of the inset shown in the upper panel. (C) OCIAD2 is an integral membrane protein. Sodium carbonate extraction fractions (pH 10.5–11.5) immunoblotted with anti-OCIAD1, anti-OCIAD2, anti-TIM50, anti-ATP5A1, and anti-SDHA antibodies. P and S indicate pellet and soluble fractions, respectively. This panel, without the OCIAD2 blot, was shown in Figure 3C. (E) OCIAD2 localizes to the inner membrane. Protease protection assay fractions immunoblotted with anti-OCIAD1, anti-OCIAD2, anti-prohibitin 2 (PHB2), anti-TIM50, anti-ATP5A1, and anti-SDHA antibodies (OMM: outer mitochondrial membrane, IMM: inner mitochondrial membrane, IMS: intermembrane space). This panel, without the OCIAD2 blot, was shown in Figure 3D. (F) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) of lauryl maltose neopentyl glycol (LMNG) detergent-solubilized mitochondrial membranes isolated from U2OS cells and immunoblotted with anti-OCIAD2 and anti-prohibitin antibodies.