Fig. 3

HRD1 targets Vimentin for polyubiquitination-mediated degradation. a Immunoblotting showing the expression of N-Cadherin and Vimentin in control cells and HRD1-overexpressing MDA-MB-231 and BT549 cell clones (#1-#3) with β-actin as a loading control. b Agarose gel electrophoresis of RT-PCR products showing the mRNA expression of HRD1 and Vimentin in control and HRD1-overexpressing MDA-MB-231 and BT549 cells. Transcript of β-actin was used as a loading control. c Vimentin mRNA level in MDA-MB-231 and BT549 cells before and after HRD1 overexpression, examined with real-time quantitative RT-PCR. β-actin was used as an internal control. d Stability tests of Vimentin in control and HRD1-overexpressing MDA-MB-231 cells treated with 50 μg/ml of cycloheximide (CHX) for the indicated times. β-actin was used as a loading control. e Immunoblotting showing the expression of Vimentin in control and HRD1-overexpression MDA-MB-231 and BT549 cells pretreated with MG132 (10 μM) or Bafilomycin A1 (BafA1, 100 nM) for 6 h. β-actin was used as a loading control. f Endogenous interaction between HRD1 and Vimentin detected by in vitro co-immunoprecipitation assay in HEK293 cells cotransfected with Myc-HRD1 and Flag-Vimentin plasmids upon MG132 (10 μM) treatment. Whole-cell lysates were immunoprecipitated with anti-Myc antibody, then immunoblotted and hybridized with the indicated antibodies. g Immunofluorescence staining showing a co-localization of HRD1 and Vimentin in the cytoplasm of MDA-MB-231 cells transfected with Myc-HRD1 plasmids upon MG132 (10 μM) treatment. Fluorophore-conjugated secondary antibodies were used as green indicating HRD1 and red indicating Vimentin. DAPI was used for staining the nucleus. Images were taken with a laser scanning confocal microscope. Scale bar = 20 μm. h Ubiquitination of Vimentin in MDA-MB-231 cells transfected with HA-ubiquitin, Myc-HRD1 or Flag-Vimentin plasmids upon MG132 (10 μM) treatment. Whole-cell lysates were immunoprecipitated with anti-Flag antibody, then immunoblotted and hybridized with the indicated antibodies. i Whole-cell lysates of HEK293 cells cotransfected with Flag-Vimentin and Myc-HRD1 or Myc-HRD1 (C291S) plasmids upon MG132 (10 μM) treatment were immunoprecipitated with anti-Flag antibody, showing the interaction between Vimentin and wild-type HRD1 or C291S mutant. j Immunoblotting showing the expression of Vimentin in MDA-MB-231 and BT549 cells before and after the overexpression of wild-type HRD1 or C291S mutant. k Stability tests of Vimentin in MDA-MB-231 cells overexpressing wild-type HRD1 or C291 mutant, treated with CHX (50 μg/ml) for the indicated times. l In vitro ubiquitination assay of Vimentin similar to Fig. 3h, with the addition of HRD1 (C291S) overexpression