Figure 4.

Phosphorylation of Y845 is not essential for EGFR autokinase activity. COS-7 cells were transiently transfected with either vector alone (−), or plasmids encoding Y845F or wt EGFR and stimulated with EGF for 5 min. The EGFR was immunoprecipitated from extracts and subjected to an in vitro kinase assay for 0 or 20 min. The reaction was stopped with the addition of sample buffer, and the products were resolved by SDS/PAGE and transferred to a membrane. After autoradiography, EGFR was detected by Western immunoblotting and visualized by using enhanced chemiluminescence (ECL; Amersham).