Mfn1 is ubiquitylated by MARCH5 at G
phase. (A) Cells were transfected with pcDNA or Myc-MARCH5 WT, and exposed to nocodazole. Myc-MARCH5 WT-expressing cells were incubated with MG132. Lysates were analyzed by immunoblotting with the indicated antibodies. (B) Cells were co-transfected with HA-ub. GFP-Mfn1 with pcDNA or Myc-MARCH5 WT. G2/M-arrested cells were obtained by treatment with nocodazole and then treated with MG132. Lysates were immunoprecipitated with an anti-GFP antibody for ubiquitylation assays. GFP-Mfn1 ubiquitylation levels were evaluated with an anti-ub antibody. (C) Cells were co-transfected with HA-ub and pcDNA, Mcy-MARCH5 WT or Myc-MARCH5H43W, after which cells were synchronized at G2/M by treatment with nocodazole with MG132. The ubiquitylation levels of endogenous Mfn1 were assayed by immunoblotting.