Fig. 4
From: Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation
Endogenous RAS phosphorylation is regulated temporally by Src and SHP2. a WT, Src/Yes/Fyn (SYF)−/− and CRISPR/Cas9-mediated SHP2−/− MEFs were serum starved and treated with 20 ng/ml of PDGF-BB. Equal amounts of lysates were immunoprecipitated with isotype-matched antibody or anti-pTyr antibody and immunoblotted with the indicated antibodies. b SYF−/− and SHP2−/− MEFs were serum starved and treated with 20 ng/ml of PDGF-BB. Equal amounts of lysates were immunoprecipitated with isotype matched antibody or anti-RAS antibody and immunoblotted with the indicated antibodies. c MEFs were serum starved and treated with 20 ng/ml of PDGF-BB or were left untreated for the indicated periods of time. Equal amounts of lysates were immunoprecipitated with isotype-matched antibody or anti-pTyr antibody and immunoblotted with the indicated antibodies. d SYF−/− and SHP2−/− MEF lysates were immunoprecipitated with isotype-matched antibody or anti-RAS antibody and analyzed by 50 μM Mn2+-PhosTag SDS-PAGE. Asterisk indicates non-specific band. e CRISPR/Cas9-mediated non-target (Ctrl) or SHP2−/− CFPAC1 cells were serum starved and treated with (+) or without (−) 10 ng/ml of EGF. Equal amounts of lysates were immunoprecipitated and immunoblotted with the indicated antibodies. f OCIP.343 xenograft tumor lysates from the indicated treatment groups were immunoprecipitated with isotype-matched antibody or anti-RAS antibody and analyzed by Mn2+-PhosTag SDS-PAGE. Arrow indicates a slower migrating band. The immunoblot data are representative of at least three independent experiments
