Vpr-DCAF interaction is not sufficient for Vpr association with DDB1. (A) 293 T cells were cotransfected with HA-Vpr or HA-VprL64P and Flag-DCAF1 expression vectors. Vpr was immunoprecipitated with anti-HA antibody and the immunoprecipitates were subjected to immunoblot analysis with anti-Flag MAb, anti-DDB1 antibody, and anti-HA antibody. (B) HA-Vpr, HA-VprR90K, or HA-VprR90D were expressed with Flag-DCAF1 and were immunoprecipitated with anti-HA antibody. Coimmunoprecipitated DDB1 and Flag-DCAF1 were detected on the immunoblot. (C) 293 T cells were transfected with increasing amounts (0.05 μg, 0.1 μg, and 0.2 μg) of HA-Vpr, HA-VprR90K, or HA-VprR90D expression vector together with a constant amount of HA-UNG2 and Flag-DCAF1 expression vectors. Two days later, the cells were lysed and Vpr and UNG2 were detected by immunoblot analysis with anti-HA antibody. The βtubulin was detected as a loading control. (D) UNG2 band intensities obtained in (C) were quantified and normalized to the UNG2 signal of the third lane from the left in (C). The results are representative data of three independent experiments.