Fig. 3. Characterization of hFis1. (A) hFis1 is located on the mitochondrial outer membrane. Samples of mitochondria (lane 1) and mitochondrial outer membrane vesicles (OMV; increased loading, lanes 2-5) were subjected to SDS-PAGE and western blot analysis using antibodies against the matrix located mtHsp70, the inner membrane complex I subunit NDUFA9, the outer membrane marker Tom40 and hFis1. (B) hFis1 is ubiquitously expressed. Isolated mitochondria from heart, brain, liver, testes, spleen and kidney rat tissues were subjected to SDS-PAGE and western blot analysis using Tom22 and hFis1 antibodies. (C) hFis1 displays a diffuse distribution along the mitochondrial surface. COS-7 cells were subjected to immunofluorescence assays using pre-immune serum, affinity-purified anti-hFis1 antibodies or mtHsp70 antibodies. (D) hFis1 is a mitochondrial protein with its N-terminus exposed to the cytosol. Isolated HeLa cell mitochondria (50 µg) were treated with or without externally added protease as indicated. Mitochondria were reisolated and subjected to SDS-PAGE and western blot analysis using mtHsp70, cytochrome c, Tom22 and hFis1 antibodies. (E) hFis1 is an integral membrane protein. HeLa cell mitochondria (100 µg) were extracted with 100 mM sodium carbonate, pH 11.5. After centrifugation at 18,000 g, the membrane pellet fraction (P; lane 1), and soluble supernatant fraction (S; lane 2) were subjected to SDS-PAGE and western analysis using antibodies directed against hFis1, mtHsp70 or Tom40.