Fig. 2. Mitofusin 2 is a ubiquitous mitochondrial protein. (A) Domain organization of mitofusin 2 (Mfn2) and of Mfn2-fragments (NG, CT) used for antibody generation. GB, GTP-binding; CC1, CC2, coiled-coil; TM, transmembrane domain; H, 6His-tag. (B) Western blots of subcellular fractions from mouse C2 and human Hela cells: TH, total homogenate; Mit, mitochondria; Cyt, cytosol; Mic, microsomes. The Mfn2-specific antiserum decorates a polypeptide (arrowhead) that is highly enriched in mitochondria, and whose apparent molecular mass is similar to that predicted for Hs-Mfn2 (86.4 kDa). The distribution of the adenine nucleotide translocator (ANT) confirms mitochondrial enrichment. The position and size (in kDa) of molecular mass markers is indicated. (C) Western blot analysis of mitochondrial fractions from human HeLa (He) and mouse C2 cells (C2) and from rat liver (L), kidney (K), brain (B) and muscle (M) with affinity purified antibodies specific for Mfn2-fragments NG or CT. (D) Double immunofluorescence on human skin fibroblasts with antibodies against the mitochondrial marker COX4 and against an N-terminal fragment (NG) of Mfn2. Mfn2 and COX4 colocalize in mitochondria. Bar, 15 µm.