FIGURE 3.

Vpr relies on the Cul4-DDB1[VprBP] E3 ubiquitin ligase to deplete MCM10. A, HeLa-iMock, HeLa-iFlag-Vpr, and HeLa-iFlag-Q65R were treated with doxycycline only for 10 h to have enough MCM10 for immunoprecipitation (IP) before it was degraded. The induced cells were harvested and lysed. The lysates were immunoprecipitated with anti-FLAG antibody conjugated to agarose beads, and the pulled down complexes were eluted with FLAG peptides. The eluates were analyzed using Western blot. B, HeLa-iMock and HeLa-iFlag-Vpr were transfected with siRNA against VprBP or non-targeting siRNA (NT). After 48 h, expression of Vpr in transfected cells was induced by doxycycline treatment for 10 h, and then cells were lysed and immunoprecipitated with anti-FLAG antibody conjugated to agarose beads. C, HeLa-iMock and HeLa-iFlag-Vpr were transfected with siRNA against VprBP or non-targeting siRNA (NT). After 48 h, expression of Vpr in transfected cells was induced by doxycycline treatment. Ten hours post-induction, the induced cells were treated with cycloheximide and harvested at 2-h intervals as indicated. Cells were lysed and analyzed using Western blot. The experiment was repeated 3 times, and one representative Western blot is shown. CHX, cycloheximide. D, VprBP binds MCM10 independently of Vpr. HeLa cells were lysed and immunoprecipitated with anti-VprBP antibody- or IgG-conjugated Sepharose beads. The pulled down proteins were released by treating the Sepharose beads with 0.1 m glycine, pH 2.0, and the eluates were then analyzed using Western blot. E, MCM10 binds VprBP and DDB1 independently of Vpr. HeLa cells were lysed and immunoprecipitated with anti-MCM10- or IgG-conjugated Sepharose beads. All experiments were repeated at least 2 times.