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. Author manuscript; available in PMC: 2019 Dec 6.
Published in final edited form as: Mol Cell. 2018 Nov 8;72(5):823–835.e5. doi: 10.1016/j.molcel.2018.09.019

Figure 3: E6 cooperates with USP46 to stabilize Cdt2 via deubiquitination.

Figure 3:

(A) Half-life measurement of Cdt2 in HeLa cells upon 18E6 or USP46 silencing: HeLa cell lysates after incubation with cycloheximide for indicated hrs were immunoblotted. Because si18E6 or siUSP46 decrease the Cdt2 protein, a longer exposure was used for Cdt2 immunoblot after si18E6 or siUSP46 so as to equalize the Cdt2 signal at 0 hr to that after siGL2. p53 induction shows si18E6 knocked down E6 effectively. (B) Co-silencing FbxO11E3 ligase stabilizes Cdt2 in HeLa cells where E6 or US46 have been knocked down. HeLa cells were transfected with indicated siRNA and lysates immunoblotted. (C) Silencing USP46 or E6 in HeLa cells increases polyubiquitination of Cdt2. HeLa cells stably expressing Flag-Cdt2 were transfected with indicated siRNAs. Cells were transfected with HA-Ubiquitin plasmid at 8hr and harvested at 48 hr. Cells treated with MG132 for 6 hrs before harvesting. Flag-Cdt2 immunoprecipitates were immunoblotted with anti-HA and anti-Flag (top) or cell lysates with indicated antibodies (bottom). (D) Knocking down E6 or USP46 increases ubiquitination of endogenous Cdt2 in HeLa cells. Same as in (C), except that endogenous Cdt2 and ubiquitin were monitored without any overexpression. (E) E6 decreases polyubiquitination of Cdt2 but this requires USP46. 293T cells transfected with indicated plasmids and siRNAs were treated with 10μM of MG132 for 8 hrs before harvest at 48 hr of plasmid transfection. Flag-Cdt2 immunoprecipitates immunoblotted with anti-HA and anti-Flag (Top 2 panels) or cell lysates immunoblotted with indicated antibodies (Bottom 3 panels). (F) as in (C) (top) and overexpression of transfected proteins detected by immunoblotting cell lysates (bottom). USP46 (C44S): catalytically dead USP46. (G) Ubiquitinated Cdt2 purified by anti-Flag antibody was incubated with myc-16E6 or IgG immunoprecipitates in presence or absence of recombinant USP46 protein and deubiquitination activity checked by western blotting with HA antibody recognizing HA-ubiquitin. (H) Separate experiment similar to Fig. 3G except that the reaction mixes were immunoblotted with anti-Cdt2 antibody and exposed to see the smear above unubiqutinated Cdt2, which represents poly-ubiquitinated Cdt2.