Cross-linking of [¹²⁵I]Tat to monocytes and effect of rTat on tyrosine autophosphorylation of the VEGFR-1/Flt-1. (A) Monocytes (2 × 10⁷) were incubated at 22°C in 0.5 mL of binding buffer with 0.5 nmol/L [¹²⁵I]Tat with or without 50-fold excess of unlabeled rTat or VEGF-A₁₆₅ for 90 minutes and then cross-linked with 1 mmol/L disuccimidyl suberate for 30 minutes. Solubilized proteins were separated by denaturating SDS-PAGE (8%) and visualized by autoradiography. (B) Human monocytes (1 × 10⁷) were stimulated with rTat (10 ng/mL) in RPMI 1640 medium containing 1 mmol/L Na orthovanadate at 4°C. Cell lysates were immunoprecipitated with antiphosphotyrosine monoclonal antibody. After SDS-PAGE (8%) separation, proteins were immunoblotted with an anti–VEGFR-1/Flt-1 antibody and visualized by enhanced chemiluminescence technique. Each experiment was repeated at least two times.