HIV-1 Tat is phosphorylated in cultured cells. HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 3, 4, 6 and 7). Lanes 1, 2, and 5 – control uninfected cells. At 48 hours post infection cells were labeled with (32P)-orthophosphate for 2 hours without (lanes 1, 3 and 6) or with (lanes 2, 4, 5 and 7) 1 μM okadaic acid (OA). Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Tat rabbit polyclonal antibodies (lanes 1–4) or anti-Flag monoclonal murine antibodies (lanes 5–7). Immunoprecipitated Tat was resolved by 15% Tris-Tricine SDS-PAGE, and transferred to polyvinylidene fluoride membrane. A, immunoblot of the membrane with anti-Tat monoclonal antibodies using the 3,3'-diaminobenzidine enhancer system. B, autoradiography of the membrane on Phosphor Imager. C, quantification of panel B. The position of light chain of IgG recognized in anti-Flag immunoprecipitates by anti-mouse HRP-conjugated secondary antibodies is indicated by asterisk.