UHRF1 catalyzes the polyubiquitination of EG5 at the site of K1034. (A) HEK293T cells were cotransfected with control or UHRF1 siRNAs, together with HA-ubiquitin and EG5-Flag. The cells then were synchronized at the G2/M phase with a double thymidine block. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with HA antibody. (B) HEK293T cells were cotransfected with plasmids expressing UHRF1-His, HA-ubiquitin, and EG5-Flag, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with an HA antibody. (C) HEK293T cells were cotransfected with plasmids expressing UHRF1-His or UHRF1ΔRING-His, HA-ubiquitin, and EG5-Flag, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with an HA antibody. (D) HEK293T cells were cotransfected with control or UHRF1 siRNAs together with HA-ubiquitin and EG5-Flag, and then the cells were synchronized in the G1/S phase using double thymidine blocking and released by culture media. The cells were synchronized in G2/M phases at 9 h after release. EG5 protein was immunoprecipitated with an anti-Flag antibody and the ubiquitination level of EG5 was assessed with HA antibody. (E and G) HEK293T cells were cotransfected with control or UHRF1 siRNAs, together with HA-K48 or HA-K63 and EG5-Flag. The cells then were synchronized at the G2/M phase with a double thymidine block. EG5 protein was immunoprecipitated with an anti-Flag antibody, and the ubiquitination level of EG5 was assessed with an HA antibody. (F and H) HEK293T cells were cotransfected with plasmids expressing UHRF1-His, HA-K48/HA-K63, and EG5-Flag, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with an anti-Flag antibody, and the ubiquitination level of EG5 was assessed with an HA antibody. (I) In vitro ubiquitination assay was performed in the presence of Ub (WT, K48, or K63), E1, E2, EG5, and UHRF1 (WT or ΔRING mutant). The ubiquitination of EG5 was examined with HA antibody. (J) HEK293T cells were cotransfected with plasmids expressing HA-ubiquitin and EG5-Flag with four lysine mutations as shown, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody, and the ubiquitination level of EG5 was assessed with an HA antibody. (K) HEK293T cells were cotransfected with plasmids expressing UHRF1ΔRING-His, HA-ubiquitin, and EG5-Flag with wild type or lysine mutation as shown, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with an HA antibody. Source data are available for this figure: SourceData F4.
Figure 4.

UHRF1 catalyzes the polyubiquitination of EG5 at the site of K1034. (A) HEK293T cells were cotransfected with control or UHRF1 siRNAs, together with HA-ubiquitin and EG5-Flag. The cells then were synchronized at the G2/M phase with a double thymidine block. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with HA antibody. (B) HEK293T cells were cotransfected with plasmids expressing UHRF1-His, HA-ubiquitin, and EG5-Flag, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with an HA antibody. (C) HEK293T cells were cotransfected with plasmids expressing UHRF1-His or UHRF1ΔRING-His, HA-ubiquitin, and EG5-Flag, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with an HA antibody. (D) HEK293T cells were cotransfected with control or UHRF1 siRNAs together with HA-ubiquitin and EG5-Flag, and then the cells were synchronized in the G1/S phase using double thymidine blocking and released by culture media. The cells were synchronized in G2/M phases at 9 h after release. EG5 protein was immunoprecipitated with an anti-Flag antibody and the ubiquitination level of EG5 was assessed with HA antibody. (E and G) HEK293T cells were cotransfected with control or UHRF1 siRNAs, together with HA-K48 or HA-K63 and EG5-Flag. The cells then were synchronized at the G2/M phase with a double thymidine block. EG5 protein was immunoprecipitated with an anti-Flag antibody, and the ubiquitination level of EG5 was assessed with an HA antibody. (F and H) HEK293T cells were cotransfected with plasmids expressing UHRF1-His, HA-K48/HA-K63, and EG5-Flag, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with an anti-Flag antibody, and the ubiquitination level of EG5 was assessed with an HA antibody. (I) In vitro ubiquitination assay was performed in the presence of Ub (WT, K48, or K63), E1, E2, EG5, and UHRF1 (WT or ΔRING mutant). The ubiquitination of EG5 was examined with HA antibody. (J) HEK293T cells were cotransfected with plasmids expressing HA-ubiquitin and EG5-Flag with four lysine mutations as shown, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody, and the ubiquitination level of EG5 was assessed with an HA antibody. (K) HEK293T cells were cotransfected with plasmids expressing UHRF1ΔRING-His, HA-ubiquitin, and EG5-Flag with wild type or lysine mutation as shown, and then the cells were synchronized at the G2/M phase. EG5 protein was immunoprecipitated with anti-Flag antibody and the ubiquitination level of EG5 was assessed with an HA antibody. Source data are available for this figure: SourceData F4.

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