WT MARCH8, but not the CS mutant, specifically ubiquitinates and downregulates TfR. WT MARCH8, but not the CS mutant, specifically ubiquitinates and downregulates TfR. HeLa cells transfected with plasmids encoding MARCH8–GFP (WT or CS mutant) for 24 hours were processed for either IF analysis with an anti-TfR antibody (A) or examined for Cy3–Tf uptake for 15 minutes (B). The cells expressing MARCH8–GFP (green in Merge) are indicated by the yellow dotted lines. The localization and expression of endogenous TfR (A) or the uptake of Cy3–Tf (B) are shown in red. Scale bar: 20 µm. (C) COS7 cells transfected with plasmids encoding GFP or MARCH8–GFP (WT or CS mutant) for 24 hours were processed for immunoblotting analysis with antibodies to GFP, TfR, and β-actin. (D) The ubiquitination of endogenous TfR in the cells expressing GFP or MARCH8–GFPs (WT or CS mutant) was examined by IP of ubiquitinated proteins with an anti-ubiquitin antibody followed by immunoblotting with an antibody to TfR. (E) The association of endogenous TfR with MARCH8 was examined by IP of MARCH8–GFP (WT or CS mutant) with an anti-GFP antibody followed by immunoblotting with an antibody to TfR. Hideaki Fujita et al. J Cell Sci 2013;126:2798-2809 © 2013. Published by The Company of Biologists Ltd