Tyrosine phosphorylation and activation of RAFTK is induced by VRP stimulation. Serum-starved HEL-JW cells were stimulated with VRP for the indicated times. TCLs were prepared and immunoprecipitated with anti-RAFTK antibody. (A) The immunoprecipitates were subjected to serial immunoblot analysis with antiphosphotyrosine antibody (upper panel) and anti-RAFTK antibody (bottom panel). The increase in tyrosine phosphorylation of RAFTK is indicated (by fold increase). (B) The immune complex was incubated with a kinase buffer containing 20 μg of poly (Glu:Tyr) (4:1) and 5 μCi γ³²P-ATP at RT for 15 minutes. The ³²P-incorporated proteins were resolved on 7.5% SDS-PAGE followed by autoradiography. Normal rabbit serum (NRS) was used as a negative control for the immunoprecipitations.