FIGURE 2.

G₂/M-defective mutants of Vpr are not able to induce proteasomal degradation of MCM10. A, HeLa cells were transduced with lentiviral vectors for expression of FLAG-tagged WT Vpr or the G₂/M-defective mutants of Vpr (Q65R and R80A) at a multiplicity of infection of 1.0. One sample was transduced with empty vector (Emp). After 40 h, cells were treated with 10 μm MG132 or DMSO, and after 8 h (total of 48 h post-transduction), cells were harvested and lysed in Laemmli buffer. Cell lysates were analyzed using Western blot. Asterisk indicates statistical significance. B, HEK293T cells were co-transfected with plasmids for the expression of HA-tagged ubiquitin (HA-UB) and FLAG-tagged WT Vpr or FLAG-tagged Q65R mutant (Q65R). After 48 h, cells were lysed and immunoprecipitated using anti-HA antibody conjugated to agarose beads. C, HeLa-iMock, HeLa-iFlag-Q65R, HeLa-iFlag-R80A, and HeLa-iFlag-Vpr cells were treated with doxycycline for 10 h and then treated with cycloheximide (CHX). Cells were then harvested and lysed at 2-h intervals as indicated. The lysates were analyzed using Western blot. The experiment was repeated 3 times, and one representative Western blot is shown.