Phosphorylation of Tks5 upon EGF stimulation requires Src. (A) COS7 cells were transiently transfected with V5-Tks5 construct and after overnight serum-starvation cells were stimulated with EGF or left untreated. Prior to stimulation, the cells were pretreated with the Src kinase inhibitors as indicated. Tks5 was then immunoprecipitated with anti-V5 antibody and subjected to anti-phosphotyrosine and anti-V5 immunoblots. (B) Serum-starved A431 cells were stimulated with EGF (50 ng/ml) for 10 min. Prior to stimulation, the cells were pretreated with the Src kinase inhibitor PP1 and the PI 3-kinase inhibitor LY294002. Endogenous Tks5 was immunoprecipitated (IP) with a polyclonal anti-Tks5 antibody. After SDS-PAGE and transfer to nitrocellulose, samples were analysed by anti-phosphotyrosine and anti-Tks5 antibodies. Cell lysates were also probed with anti-pAKT (Ser473) or anti-Akt antibodies. These results are typical of at least three experiments.