Figure 3

Tax-P79AQ81A activates the cytoplasmic steps of the NF-κB pathway. (A) Ability of wt Tax and Tax-P79AQ81A to bind to endogenous NEMO and induce the phosphorylation of IKKα/β subunits. HeLa cells were transfected with a control plasmid or with the Tax-His constructs, including the NF-κB-defective Tax-M22 mutant as a negative control. Proteins precipitated using the anti-Tax mab (IP Tax) were blotted with either an anti-NEMO antibody or with the anti-Tax sera. In parallel, total proteins (Lysates) were blotted with anti-Tax sera and with anti-NEMO or anti-phospho-IKK antibodies, as indicated. NS: non specific. (B) Purification of aggresomes formed in wt Tax or Tax-P79AQ81A producing HeLa cells. Soluble and insoluble fractions were prepared according to [37] and proteins in each fraction were blotted with either an anti-NEMO antibody or the anti-Tax sera. (C D) Confocal microscopy analysis performed in HeLa cells showing the distribution of Tax (green) and endogenous NEMO (C) or RelA (D) (red). Nuclei were stained with DAPI (blue). At least 100 cells were analyzed in each condition.