Tax C-terminal lysines are critical for IKK binding, IKK activation, and nuclear translocation of RelA. (A) HeLa cells were transfected with Tax or Tax mutants. Lysates were immunoprecipitated with anti-Tax mab and recovered proteins were blotted using anti-Tax, anti-IKKα, anti-IKKβ, or anti-IKKγ antibodies, as indicated. (B) HeLa cells were transfected as in panel A. Lysates were immunoprecipitated using anti-IKKγ antibodies and recovered proteins were blotted using anti-Tax, anti-IKKα, anti-IKKβ,or anti-IKKγ antibodies, as indicated. (C) Jurkat cells were transfected with Tax or Tax mutants. Activity of immunoprecipitated IKK was assessed by kinase assay using GST-IκB-α as substrate (top panel). Immunoprecipitates were probed with anti-IKKα antibody to determine the amounts of precipitated kinase and with anti–GST-IκB-α to determine the amount of substrate. Equal amounts of lysates were determined by blotting with anti-GAPDH antibody. (D) HeLa cells were transfected with either Tax plasmid and stained with anti-Tax and anti-RelA antibodies. The percentage of nuclear translocation of RelA in Tax-positive cells is indicated.