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Figure 5 | Retrovirology

Figure 5

From: HIV-1 Vpr stimulates NF-κB and AP-1 signaling by activating TAK1

Figure 5

Vpr increases the polyubiquitination of TAK1. (A) Vpr synergizes with TAB3 to induce the polyubiquitination of TAK1. HEK293T cells (4 × 106) were transfected with Myc-TAK1 together with Flag-Vpr, T7-TAB3 or HA-Ub. Forty-eight hours after transfection, cells were harvested and polyubiquitination of the immunoprecipitated TAK1 was detected in Western blotting with anti-HA antibody. (B) Vpr does not affect the auto-ubiquitination of TRAF6. HEK293T cells (4 × 106) were transfected with HA-TRAF6 together with Vpr or Ub DNA. Cell lysates were immunoprecipitated with anti-HA antibody. Samples from both cell lysates and immunoprecipitates were probed with indicated antibodies. (C) Vpr promotes the association between TAK1 and TAB3. A total of 1 × 107 HEK293T cells were transfected with an empty vector or Flag-Vpr DNA construct. After forty-eight hours, cells were pretreated with 20 nM Calyculin A for 5 min. Then whole cell lysates were immunoprecipitated with anti-TAK1 antibody. Samples from both cell lysates and immunoprecipitates were probed with rabbit anti-TAK1, anti-TAB1, anti-TAB2, anti-TAB3, anti-Flag, and anti-Tubulin antibodies. (D) TAB3 is involved in Vpr-induced phosphorylation of TAK1. The control and TAB3-knockdown HeLa cell lines (0.25 × 106) were transfected with 1 μg empty vector or Flag-Vpr DNA constructs. After 48 hours, cells were pretreated with 20 nM Calyculin A for 5 min before cells were collected and analyzed by Western blotting with indicated antibodies.

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