TRAF6 is required for Vpr-induced TAK1 phosphorylation. (A) Jurkat cells (2 × 106) were transduced with retroviruses expressing shRNA targeting TRAF6, TRAF2 or a scrambled shRNA (shControl). After forty-eight hours, cells were exposed to VSV-G pseudotyped WT or ΔVpr (equivalent to 500 ng p24) in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After two hours, cells were pretreated with 15 nM Calyculin A for 5 min. Whole cell lysates were examined in Western blotting with the indicated antibodies. (B) HEK293T cells (4 × 106) were co-transfected with wild type TAK1 or its mutants (K34R, K158R, K209R) with or without HA-Ub DNA constructs. Forty-eight hours after transfection, cells were collected and denatured by boiling with 1% SDS, followed by immunoprecipitation with anti-Myc antibody. Polyubiquitination of TAK1 was detected with anti-HA antibody. Samples from both cell lysates and immunoprecipitates were probed with anti-TAK1 antibody. (C) Polyubiquitination of TAK1 is required for Vpr-induced TAK1 phosphorylation. HEK293T cells (0.5 × 106) were transfected with 0.3 μg Myc-TAK1 or its mutants (K34R, K158R, K209R) along with empty vector or 0.5 μg Flag-Vpr. After forty-eight hours, whole cell lysates were subjected to western blotting and probed with indicated antibodies.