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. 2007 Jul 2;178(1):71–84. doi: 10.1083/jcb.200611064

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Copyright © 2007, The Rockefeller University Press

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Figure 7. — Expression of MARCH5 RING mutants restores tubular mitochondria in Mfn1KO and Mfn2KO cells. (A) Expression levels of endogenous Mfn2 in wild-type MARCH5 and MARCH5^H43W-expressing cells was analyzed by Western blot. The whole cell lysate (WCL) and mitochondria-enriched heavy membrane (HM) fractions were obtained from HeLa cells incubated in the presence and absence of MG132 and transfected with Myc-tagged MARCH5 and MARCH5^H43W. Proteins were resolved using SDS-PAGE and then immunostained with anti-Mfn2 polyclonal antibodies. The loading was controlled by immunostaining with anti-Hsp60 antibodies. Mfn2KO cells transfected with MARCH5-YFP (B; yellow on overlay image) or MARCH5^H43W-YFP (C and D; green on overlay images) were stained with anti-cytochrome c mAbs to reveal mitochondria (red on overlay images) and analyzed by confocal microscopy. In E, cells were scored for the morphology of mitochondria. Data represent averages ± SD of three transfections with 100 cells counted each time.