FIGURE 4.

CRL4-DCAF1 E3 ubiquitin ligase is involved in Vpr-mediated MUS81-EME1 down-regulation. A, MUS81 and EME1 plasmids were co-transfected with increasing amounts of Vpr or balanced empty vector plasmid and empty vector or DCAF1 plasmids as indicated. 48 h later, cells were harvested, and some lysates were analyzed by Western blotting with appropriate antibodies. B, MUS81 and EME1 plasmids were co-transfected with increasing amounts of DCAF1 or balanced empty vector plasmids and empty vector or Vpr plasmids. C, DCAF1 siRNA or control siRNA was transfected 24 h prior to co-transfection of Vpr, MUS81, EME1, or balanced empty vector plasmids as indicated. D, MUS81 and EME1 plasmids were co-transfected with Vpr and/or DCAF1 plasmids, which were balanced by empty vector plasmids. 42 h later, MG132 (10 μm final concentration) in DMSO or DMSO only was added to the cell culture. After 6 h, cells were harvested, and some lysates were analyzed by Western blotting. Co-immunoprecipitation (IP) with anti-FLAG antibody was performed and analyzed by immunoblotting. E, plasmids expressing MUS81, Vpr, DCAF1, and DDB1 were co-transfected into cells as indicated. Protein levels in cell lysates and co-immunoprecipitation were analyzed as described above. All experiments were repeated three times with similar results.