FIGURE 4.

CGI-58 regulates mitochondrial dynamics as a mitochondrial protein. A, subcellular fractionation analysis of CGI-58 stably expressed in C2C12 cell. Total cell lysate from C2C12 cells were subjected to differential centrifugation to separate into the crude mitochondrial fraction (crude mito), mitochondria-associated membranes (MAM), pure mitochondria (Mito), microsomal fraction, and cytosol, followed by Western blot analysis using antibodies against FLAG, calnexin, and Tom20, which were used as markers for CGI-58, endoplasmic reticulum, and mitochondria, respectively. B, confocal image analysis of mitochondrial dynamics. COS-7 cells were transiently co-transfected with expression vectors for mito-EGFP and CGI-58 or empty vector. After 48 h of transfection, the cells were analyzed for mitochondrial dynamics and the expression pattern of CGI-58 by confocal image analysis using anti-FLAG antibody and Cy3-conjugated donkey-anti-mouse IgG. Scale bar, 10 μm. C, quantitative analysis of mitochondrial morphology in COS-7 cells overexpressing CGI-58 or vector control in three categories: tubular, mix, and fragmented from three independent experiments (n = 150 for each independent experiment).