Fig. 6 | Oncogene

Fig. 6

From: Correction: The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation

Fig. 6

RNF31 triggers ERα mono-ubiquitination. a Detection of a potentially mono-ubiquitinated form of ERα upon RNF31 overexpression. HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged RNF31 or the Myc-tag alone. Forty-eight hours after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31, ERα, mono-ubiquitinated ERα and of the internal control GAPDH are indicated. b Detection of endogenous mono-ubiquitinated ERα upon RNF31 depletion. MCF-7 cells were transfected with siRNF31 or siControl. Forty-eight hours after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31, ERα, mono-ubiquitinated ERα and the internal control GAPDH are indicated. c Deletion of the RNF31 RBR domain abolishes the potentially mono-ubiquitinated form of ERα. HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged full-length RNF31 derivatives or the Myc-tag alone. Forty-eight hours post transfection, cell extracts were prepared and ERα forms were detected by western blot analysis. The predicted molecular weights of RNF31, ERα, mono-ubiquitinated ERα and of the internal control GAPDH are indicated. d Direct evidence for ERα mono-ubiquitination. Immunoprecipitation of ubiquitinated proteins from MCF-7 cell extracts upon overexpression of RNF31. Ubiquitinated ERα species were detected by western blots using anti-ERα, identifying a prominent 75 kDa mono-ubiquitinated ER form. e ERα mono-ubiquitination requires the RNF31 RBR domain. Plasmids expressing Myc-tagged RNF31 derivatives were transfected into HEK-293 cells together with the ERα expression plasmid. Whole-cell extracts were subjected to immunoprecipitation of ERα and subsequently analyzed for ubiquitinated ERα forms by western blot analysis using anti-ubiquitin. The predicted molecular weight of mono-ubiquitinated ERα is indicated