Fig. 8. Effect of binding site mutations on TrkA-Ros association with SHP-1 and dephosphorylation as revealed by biochemical methods. (A) TrkA-Ros-ECFP-wt or the respective mutants were coexpressed with catalytically inactive SHP-1CS in HEK293 cells. TrkA-Ros-ECFP was immunoprecipitated from cell lysates with anti-GFP antibodies, and associated SHP-1CS was detected with anti-SHP-1 antibodies. Control: IgG isotype precipitation. Consistent results were obtained in corresponding experiments using untagged TrkARos. (B) Dephosphorylation of TrkA-Ros-ECFP or the respective mutants by coexpressed SHP-1-wt. TrkA-Ros-ECFP-wt or the YF mutants, as indicated, were expressed in HEK293 cells with or without different amounts of SHP-1 wild-type. Lysate aliquots were analyzed with anti-phosphotyrosine antibodies (4G10) for TrkA-Ros-ECFP phosphorylation. Expression levels of receptor and SHP-1 were comparable (not shown). The resulting bands for three independent experiments with a ratio of TrkA-Ros-ECFP:SHP-1 of approximately 1:2 were quantified, normalized to TrkA-Ros-ECFP expression levels, and relative ratios of tyrosine phosphorylation in presence or absence of SHP-1 were calculated (lower panel: means±s.e.m.; ^*significantly different from wild-type, P<0.05).