PI 3-kinase and intact PX domain are required for tyrosine phosphorylation of Tks5. (A) COS7 cells were transiently transfected with V5-Tks5 and after serum-starvation cells were stimulated with EGF or left untreated. Prior to stimulation the cells were treated with the PI 3-kinase inhibitors LY294002 and BKM120, respectively. Tks5 proteins were immunoprecipitated with anti-V5 antibody and subjected to anti-phosphotyrosine and anti-V5 immunoblots. Cell lysates were also probed with anti-pAKT (Ser473) or anti-Akt antibodies. (B) COS7 cells were transiently transfected with the V5-Tks5R42A construct and challenged with EGF. Cell lysates were then subjected to immunoprecipitation with anti-V5 antibody. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine and anti-V5 antibodies. These results are typical of at least three experiments.