Fig. 4. Mfn1 co-fractionates with mitochondria and participates in a high molecular weight complex. (A) Mfn1 co-fractionated with the mitochondrial outer membrane proteins TOM40 and porin. Postnuclear protein extracts from HeLa cells and mouse heart were fractionated by differential centrifugation, subjected to SDS-PAGE, and then western blots were probed with anti-Mfn1, anti-TOM40, anti-porin and anti-actin. Total, postnuclear extract. PMS, postmitochondrial supernatent, depleted of mitochondria. Mito, crude mitochondrial pellet. (B) Migration of Mfn1 in a 350 kDa complex. Mitochondrial extracts derived from human HL-60 cells were solubilized with digitonin and fractionated through a Superose-6 gel filtration column. Fractions were analyzed by western blotting using anti-Mfn1 antibodies, void volume to the left; mobility of marker proteins (arrows).