Tax C-terminal lysines are critical for NF-κB, but not CREB, activation. (A) Schematic representation of the lysine-to-arginine Tax mutants. (B) 293T cells were transfected with the various Tax-6His plasmids and proteins purified by denaturing Ni-NTA pull-down were blotted with mabs to poly-ubiquitin chains (top panel) or to Tax (middle and bottom panels). (C) Representation of Tax mutants in which lysines 7 and/or 8 were reintroduced to the lysineless protein K1-10R-6His. (D) 293T cells were transfected with Tax-6His, the lysineless Tax K1-10R-6His, or the indicated reverse mutants in the presence of an HA-Ub plasmid to favor Tax ubiquitylation. Proteins purified by denaturing Ni-NTA pull-down were blotted with mabs to HA (top panel) or to Tax (middle and bottom panels). (E) Jurkat cells were cotransfected with the NF-κB-Luc (top panel) or the HTLV-LTR-Luc (bottom panel) reporter plasmids, and the plasmids encoding for Tax or Tax mutants. For normalization, cells were cotransfected with renilla luciferase expression plasmid. Activity of the wild-type protein was set to 100%. The results represent the mean plus or minus standard deviation of at least 3 different experiments. (F) Jurkat cells were cotransfected with Tax or Tax mutants. NF-κB DNA-binding activity was assessed by electrophoretic mobility shift assay using a consensus oligonucleotide for NF-κB. Specificity of the NF-κB complex was determined by the addition of an excess of an unlabeled consensus (cold) or mutated (mutant) oligonucleotides.