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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 6. — MARCH5 RING mutations decrease cellular mobility of Drp1. HeLa cells transiently transfected with YFP-Drp1 with or without cotransfection with MARCH5-CFP, MARCH5^H43W-CFP, and MARCH5^C65S,C68S-CFP were analyzed for cellular mobility of Drp1 with the FRAP assay (A). The averaged recovery curves and the FRAP values quantified at 1.73 s and 19.66 s after photobleach in the indicated experimental groups are shown at the bottom (A). The degree of Drp1 association with mitochondria was analyzed by Western blot (B). The whole cell lysate (WCL) and mitochondria- enriched heavy membrane (HM) fractions were obtained from HeLa cells transfected with Myc-tagged, wild-type MARCH5 and MARCH5^H43W. Proteins were resolved using SDS-PAGE and then immunostained for Drp1. The loading was controlled with anti-Hsp60 antibodies. The HM fractions obtained from cells expressing Myc-tagged, wild-type MARCH5, and MARCH5^H43W were also treated with a chemical cross-linker BMH, or with solvent DMSO. The samples were analyzed by Western blot for Drp1 (C).