Fig. 7: RNF208 degrades the soluble form of Vimentin by recognizing the phosphorylation of Vimentin at the Ser39 residue.

a Immunoblot analysis of the expression levels of Vimentin in soluble and insoluble fractions isolated from control and RNF208-overexpressing MDA-MB-231 cells. b The 293T cells were cotransfected with Myc-RNF208 and Flag-Vimentin mutant (wild-type, S39A, S56A, S73A, S83A) plasmids upon MG132 treatment. Cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with the indicated antibodies. c Immunoprecipitation assay showing the endogenous interaction between RNF208 and phosphorylated Vimentin at the Ser39 residue in RNF208-overexpressing MDA-MB-231 cells with or without MG132. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. d The 293T cells were cotransfected with wild-type, inactive mutant (S39A), or constitutively active mutant (S39D) of Flag-Vimentin in the absence or presence of Myc-RNF208. Cell lysates were subjected to immunoblotting with the indicated antibodies. e After Flag-Vimentin WT, S39A, or S39D plasmids were cotransfected into 293T cells with or without Myc-RNF208 plasmid upon MG132 treatment, cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. f HA-Ubiquitin plasmid was cotransfected into 293T cells with wild-type, S39A, or S39D plasmids of Flag-Vimentin in the absence or presence of Myc-RNF208 plasmid upon MG132 treatment. Cells lysates were immunoprecipitated with anti-Flag antibody, and ubiquitinated Vimentin was observed by immunoblotting using an anti-HA antibody. Unprocessed original scans of blots in (a−f) are shown in Supplementary Fig. 13.