p13 competes with p300 for Tax interaction. (A) 293T cells were transfected with (+) or without (−) Tax and with increasing amounts of p13-HA. Tax-normalized lysates were immunoprecipitated using anti-Tax antibody and immunoblotted with anti-p300 and anti-Tax antibodies. The right panel shows immunoblots of total cell lysates (input) with anti-HA and anti-p300 antibodies. (B) 293T cells were transfected with empty vector, without (−) and with (+) pcTax and increasing amounts of p13-HA and with and without p300-Flag (5 μg). Lysates were immunoprecipitated with anti-p300 antibody and both immunoprecipitate and input (right panel) were immunoblotted with anti-HA, anti-Tax, and anti-Flag. (C) 293T cells were transfected with empty vector, p13-HA without (−) and with (+) Tax, or Tax alone. Cell lysates were immunoprecipitated using anti-Tax antibody. Immunoprecipitates (left panel) and total cell lysates (input, right panel) were immunoblotted with anti-HA and anti-Tax antibodies. (D) In vitro association of Tax and p13. In vitro transcribed and translated³⁵  S-methionine–labeled p13 protein was incubated with GST (top panel lane 2) or GST-Tax (top panel lane 3) immobilized on glutathione-agarose beads. Radiolabeled p13 bound to GST-Tax was visualized with a PhosphorImager. The bottom panel is a Coomassie blue staining of the same gel as in the top panel showing GST (lane 1) and GST-Tax (lane 2) protein present in each reaction. (E) 293T cells were transfected with vector control, Tax without and with increasing amounts of p300, or Tax with p13 and increasing amounts of p300. The reporter construct HTLV-1 LTR-Luc was included and luciferase activity adjusted for transfection efficiency using RSV-βgal. DNA concentrations were equalized using the appropriate backbone vectors. SD is given for the average of 3 independent experiments. Lysates were examined by immunoblotting for expression of Tax, Flag-p300, and tubulin.