FIG. 3.
Lysines located in the C-terminal half of Tax are the principal targets of ubiquitin. (A) Schematic representation of the lysine-to-arginine Tax mutants. (B) 293T cells were transfected with the control plasmid pSG5 (lane 1) or with Tax-6His (lane 2), Tax-6His K1-10R (lane 3), Tax-6His K1-3R (lane 4), Tax-6His K4-10R (lane 5), or Tax-6His K6-8R (lane 6). Proteins eluted after Ni-NTA pulldown were subjected to Western blot analysis with a rabbit polyclonal serum to Tax (left panel) or the anti-ubiquitin Fk2 monoclonal antibody (right panel). (C) Quantitation performed on the anti-ubiquitin blot after subtraction of the background in lane 1 and normalization to an equal amount of the unmodified 40-kDa species.