The degree of Tax NF-κB activity correlates with the levels of Tax ubiquitination but not SUMOylation. (A) NiNTA experiments performed in 293 T cells showing the levels of Tax conjugation to endogenous ubiquitin or SUMO. 293 T cells were transfected as indicated and proteins purified on Nickel columns were revealed using anti-Ubiquitin, anti-SUMO-2/3 or the anti-Tax sera. The percentages of ubiquitinated or SUMOylated Tax were normalized on the amount of unconjugated Tax and expressed in comparison to wt Tax (100%). (B) NF-κB reporter assay in 293 T cells. Cells were transfected with control or Tax-His constructs along with the NF-κB or the Renilla luciferase plasmid for normalization. Fold induction was calculated by dividing the firefly/renilla ratio of each Tax protein with the ratio of the control plasmid. Results represent the means and standard error of the means (SEM) from three independent experiments performed in duplicates. (C) The level of either ubiquitination or SUMOylation of the Tax mutants was plotted against their NF-κB activities. The regression line shows a correlation between Tax NF-κB activity and ubiquitination (determination coefficient = 0.94) but not SUMOylation (determination coefficient = 0.73). (D) Association of the Tax proteins to the IKK complex in 293 T cells. Total proteins (Lysates) or proteins precipitated using the anti-Tax mab (IP Tax) were blotted with anti-NEMO, anti-phospho-IKK or the anti-Tax sera, as indicated. (E) The regression line shows a correlation between Tax binding to phospho-IKKα/β and ubiquitination (determination coefficient = 0.94) but not SUMOylation (determination coefficient = 0.58).