Effects of mutations of the C-terminal tyrosines of LAT on the tyrosine phosphorylations of PLCγ2 in response to Cvx. (A,B) Platelets from WT, Lat3YF, and LatY136F mice were stimulated by 5 nM Cvx for the time indicated. Reactions were stopped by addition of RIPA buffer, protein separated by a 12.5% SDS-PAGE, and transferred onto nitrocellulose; the tyrosine-phosphorylated proteins were detected by immunoblotting with the antiphosphotyrosine antibody 4G10. The membrane was stripped and reprobed for LAT, Syk, SLP-76, and PLCγ2; their positions are indicated by the black arrow (from the bottom of the nitrocellulose). Platelets from WT (C,D) (left panel), Lat3YF (C) (right panel), and LatY136F (D) (right panel) mice were stimulated by 5 nM Cvx for the time indicated. Reactions were stopped by addition of RIPA buffer, and LAT was immunoprecipitated using a specific antibody. Tyrosine phosphorylation of LAT was assessed by immunoblotting using the antiphosphotyrosine antibody 4G10 (top panels). The membrane was stripped and reprobed for LAT with the anti-LAT antibody (bottom panels). PLCγ2 was immunoprecipitated from the lysate of platelets from WT (E,F) (left panel), Lat3YF (E) (right panel), and LatY136F (F) (right panel) mice stimulated by 5 nM Cvx for the indicated times. Tyrosine phosphorylation of PLCγ2 was assessed by immunoblotting using the antiphosphotyrosine antibody 4G10 (top panels). The membrane was stripped and reprobed for PLCγ2 with the anti-PLCγ2 antibody (bottom panels). Results are representative of 3 independent experiments.