Figure 6

TAK1 is required for Vpr-induced activation of NF-κB and AP-1 signaling. (A-B) Vpr promotes TAK1-mediated phosphorylation of IKKβ and MKK7. HEK293T (0.5 × 10⁶) were transfected with 0.3 μg HA-IKKβ (A) or 0.5 μg HA-MKK7 (B), with or without Myc-TAK1 and Flag-Vpr DNA constructs. After forty-eight hours, whole cell lysates were subjected to Western blotting and probed with indicated antibodies. (C) Expression of TAK1, TAB3, and TRAF6 in HeLa knockdown cell lines. Retroviruses coding shControl, TAK1, TAB3, or TRAF6 shRNA sequences, were stably transduced into HeLa cells. Cell lysates were immunoblotted with indicated antibodies. (D-F) Endogenous TAK1, TRAF6, and TAB3 are required for activation of NF-κB, AP-1, and HIV-1 LTR by Vpr. The aforementioned HeLa knockdown cell lines (0.1 × 10⁶) were transfected with vector or Flag-Vpr along with κB (D), AP-1 (E), or HIV-1 LTR (F) luciferase reporter plasmids. After forty-eight hours, luciferase activities were measured. The activation fold by Vpr was calculated by dividing the luciferase activity from Vpr-transfected cells by the luciferase activity from vector-transfected cells. The results shown are the averages of three independent experiments. The error bars indicate standard deviations. *P < 0.05, **P < 0.01 (paired t test).