Figure 3: SHP2 binds to tyrosyl phosphorylated Ras in the presence of Src.

(a) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (b) Wild-type MEFs or Src/Yes/Fyn triple-knockout (SYF−/−) MEFs were serum starved and pretreated with (+) or without (−) II-B08 and then treated with (+) or without (−) 1 ng ml⁻¹ of EGF. Equal amounts of lysates were immunoprecipitated with anti-SHP2 antibody and immunoblotted with the indicated antibodies. (c) U87 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by a treatment with (+) or without (−) 1 ng ml⁻¹ of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. (d) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) indicated concentration of II-B08 followed by treatment with (+) or without (−) 1 ng ml⁻¹ of EGF, lysed, immunoprecipitated with control rabbit IgG or anti-HA antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.