Fig. 6: Activity of the RNF208 E3 ligase is required for the polyubiquitination-mediated degradation of Vimentin through interaction with its head domain. | Nature Communications

Fig. 6: Activity of the RNF208 E3 ligase is required for the polyubiquitination-mediated degradation of Vimentin through interaction with its head domain.

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

Fig. 6

a Wild-type RNF208 or RNA208 mutant (C143A, C167A, C186A) plasmids were cotransfected with Flag-Vimentin into 293T cells upon MG123 treatment. b After the Flag-Vimentin plasmid was cotransfected with the HA-Ubiquitin plasmid into 293T cells with wild-type RNF208 or RNF208 mutant (3MT, C143A/C167A/C186A), cell lysates were immunoprecipitated with anti-Flag antibody, and ubiquitinated Vimentin was observed by immunoblotting using an anti-HA antibody. c Immunoblot analysis showing the expression levels of Vimentin in control, wild-type RNF208, and RNF208 mutant (3MT)-overexpressing MDA-MB-231 cells. d HA-Ubiquitin plasmid was cotransfected into 293T cells with wild-type or lysine mutant (K97A) of Flag-Vimentin in the absence or presence of Myc-RNF208 plasmid. Cell lysates were immunoprecipitated with anti-Flag antibody, and ubiquitinated Vimentin was observed by immunoblotting using anti-HA antibody. e, f Flag-Vimentin or Flag-Vimentin (K97A) plasmids were cotransfected into 293T cells in the absence or presence of Myc-RNF208 plasmids. Cells were treated with cycloheximide (CHX, 50 μg ml−1) for the indicated times (e). The data were quantified using ImageJ software (f), and β-actin was used as an internal control. g Representative whole lung image stained with India ink showing metastatic nodules from 8 weeks, derived from lateral tail-vein injection of control, wild-type RNF208 or RNF208 mutant (3MT)-overexpressing MDA-MB-231 cells (left) (n = 5 per group). Scatter plot showing the number of lung metastatic nodules (right). The P value was calculated by unpaired two-tailed Student’s t tests. The data represent the mean ± SD. h Representative IHC image showing RNF208 and Vimentin expression in lung tissues from (g). Original magnification ×100. Scale bar, 50 μm. Source data for (g) are available in Source Data file. Unprocessed original scans of blots in (a−e) are shown in Supplementary Fig. 13.

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