UHRF1 catalyzes the K63-linked ubiquitination of EG5 at K1034. (A and B) HEK293T cells were cotransfected with plasmids expressing UHRF1-His or UHRF1^ΔRING-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. (C) HEK293T cells were cotransfected with plasmids expressing HA-K63 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 an anti-Flag antibody, and the ubiquitination level of EG5 was assessed with an HA antibody. (D) HEK293T cells were cotransfected with plasmids expressing UHRF1^ΔRING-His, HA-K63, 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 FS2.