Expression of untagged and Flag-tagged Tat. COS-7 cells were transfected with Tat (lane 3) and Flag-tagged Tat (lane 4) expression vectors or mock-transfected (lane 2). At 48 hours post transfection cells were lysed and Tat was immediately immunoprecipitated with anti-Tat rabbit polyclonal antibodies (lanes 2–4). Immunoprecipitated Tat was resolved by 15% Tris-Tricine SDS-PAGE, transferred to polyvinylidene fluoride membrane and immunoblotted with anti-Tat monoclonal antibodies using the 3,3'-diaminobenzidine enhancer system. Positions of Tat and Flag-Tat are indicated. Lane 1, prestained 10 kDa molecular weight markers.