Fig. 4: RNF208 induces the proteasomal degradation of Vimentin protein by facilitating K27-linked polyubiquitination. | Nature Communications

Fig. 4: RNF208 induces the proteasomal degradation of Vimentin protein by facilitating K27-linked polyubiquitination.

From: RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers

Fig. 4

a List of RNF208 binding partner candidates analyzed from the mass spectrometry-based crosslinking assay. b Immunoblot analysis showing Vimentin, E-cadherin, and RNF208 in control and RNF208-overexpressing Hs578T and MDA-MB-231 clonal cells (#1−#3). β-actin was used as an internal control. c RT-PCR showing RNF208 and VIM expression in control and RNF208-overexpressing Hs578T or MDA-MB-231 cells; 18S was used as an internal control. d Immunoblot analysis showing the stability of the Vimentin protein in control and RNF208-overexpressing MDA-MB-231 cells in the presence of cycloheximide (CHX, 50 μg ml−1) for the indicated times (left). The data were quantified using ImageJ software (right), and β-actin expression was used for normalization. e Immunoblot analysis showing the expression level of Vimentin protein in control and RNF208-overexpressing Hs578T and MDA-MB-231 cells with the proteasome inhibitor MG132. Cells were treated with 10 μM MG132 for 6 h, and cell lysates were subjected to immunoblotting with the indicated antibodies. f Immunoprecipitation assay showing the endogenous interaction between RNF208 and Vimentin. Cells were cotransfected with Myc-RNF208 and Flag-Vimentin plasmids in 293T cells upon MG132 treatment. Cell lysates were immunoprecipitated with anti-Myc antibody and then immunoblotted with the indicated antibodies. WCL whole-cell lysates. g Immunoprecipitation assay showing the ubiquitination of Vimentin by ectopic expression of RNF208. 293T cells were cotransfected with HA-Ubiquitin, Flag-Vimentin, or Myc-RNF208 plasmids in 293T cells upon MG132 treatment. Vimentin ubiquitination was detected by immunoprecipitation with anti-Flag and then immunoblotted with the indicated antibodies. h Immunoprecipitation assay showing K27-mediated ubiquitination of Vimentin by RNF208. The 293T cells were cotransfected with Flag-Vimentin, different linkages of HA-Ubiquitin (wild-type, K27, K29, K49. K63), or Myc-RNF208 plasmids, and then, cell lysates were immunoprecipitated with anti-Flag antibody. i Flag-Vimentin plasmid was cotransfected into 293T cells together with wild-type, K27 or lysine mutant (K27R) of HA-Ubiquitin in the absence or presence of Myc-RNF208 plasmid. Cells lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with the indicated antibodies. Unprocessed original scans of blots in (b−i) are shown in Supplementary Fig. 13.

Back to article page