HBx spatially isolate USP37 from its E3 ligases to inhibit USP37 ubiquitination.

(A) Representative confocal image of Huh7 cells transiently expressing HA-CDH1 or Myc-β-TrCP constructs, fixed and stained with α-CDH1 or α-β-TrCP and α-USP37 primary antibody and corresponding fluorescent-labelled secondary antibody. Nuclei were counterstained with DAPI. Images were captured at 60x magnification in Nikon A1R confocal microscope. Scale bar represents 10 µm unless mentioned otherwise. (B) Cell extracts from cells transfected with Vector, HBx-GFP and HBx-NESM-GFP as indicated, were separated on SDS-PAGE gel and western blotted with antibody against USP37. GAPDH was used as loading control. (C) Ubiquitination assay was performed by immunoprecipitating cell lysates from Huh7 cells ectopically expressing Vector and Myc-Ubi, HBx-GFP and Myc-Ubi or HBx-NESM-GFP and Myc-Ubi (2.5 ug DNA of each recombinant was to make a total of 5 ug DNA was transfected in 100 mm Dish); and treated with 20 µM MG132 for 6 h, with USP37 antibody. Immuno-complexes were eluted and western blotted with α-Ubiquitin antibody. (D) Cell extracts from Huh7 cells transiently transfected with Vector, HBx-GFP or HBx-NESM-GFP were immunoprecipitated with α-CDH1 antibody. Immuno-complexes were western blotted with α-USP37 and α-CDH1 antibodies. (E) Cell extracts from Huh7 cells transiently transfected with Vector, HBx-GFP or HBx-NESM-GFP constructs were immunoprecipitated with α-β-TrCP antibody. Immuno-complexes were western blotted with α-USP37 and α-β-TrCP antibodies. (F) Ubiquitination assay was performed by immunoprecipitating cell lysates from Huh7 cells transiently expressing Vector and Myc-Ubi; HA-CDH1 and Myc-Ubi; Myc-β-TrCP and Myc-Ubi; HA-X0, HA-CDH1 and Myc-Ubi; HA-X0, Myc-β-TrCP and Myc-Ubi or HA-X0 and Myc-Ubi (Each 100 mm dish was transfected with 2 ug DNA of indicated plasmids to make a total of 6 ug DNA transfected per dish. Where co-expression of two plasmids (total DNA-4 ug) is indicated the transfection was normalized with 2 ug of Vector construct to ensure equal transfection of DNA); and treated with 20 µM MG132 for 6 h, with USP37 antibody. Immuno-complexes were eluted and western blotted with α-Ubiquitin antibody.