Vpr enhances the phosphorylation of TAK1 in HeLa and 293T cells. (A-B) Vpr induces the phosphorylation of exogenous TAK1 in a dose-dependent manner. HeLa cells (0.25 × 106) (A) and HEK293T (0.5 × 106) (B) were transfected with 0.3 μg Myc-TAK1 and increasing amounts of Flag-Vpr, or 0.3 μg HA-TAB1. Different amounts of Vpr plasmid were transfected into HEK293T cells (0, 0.2, 0.5 and 1 μg) and HeLa cells (0, 0.5, 1, and 1.5 μg). Forty-eight hours after transfection, whole cell lysates were harvested and probed with indicated antibodies. (C) Vpr induces the phosphorylation of endogenous TAK1. HEK293T cells were transfected with 1 μg Flag-Vpr or an empty vector. After forty-eight hours, cells were pretreated with DMSO or 20 nM Calyculin A for 5 min. Whole cell lysates were subjected to Western blotting and probed with indicated antibodies.