(A) Hierarchical clustering of relative SH2 specificity. Values were obtained by subtracting percent bound by pY from percent bound by each SH2 for each time point. Red represents binding greater than pY and green represents binding less than pY. Relative SH2 specificity value = %Band intensitySH2 at time x - %Band intensityanti-pY at time x. (B) Hierarchical clustering of SH2 binding to EGFR band (normalized to maximum, max=1, min=0) for 60-min time-course. (C) Hierarchical clustering of EGFR phosphosite-specific Western blotting quantifications (normalized to maximum, max=1, min=0) for 60-min time-course. (D) and (E) Plots comparing the EGFR phosphosite kinetics and SH2 binding kinetics for SH2 domains and their canonical binding motifs (D, CRKL and pY992, R2=0.81; E, GRB2 and pY1168, R2=0.91). (F) Plot comparing total EGFR phosphorylation (pY EGFR band) with SHCA PTB binding (R2=0.89). R-squared values were calculated by plotting normalized pY Western intensity against normalized SH2 binding FW intensity. pY EGFR quantifications consist of data from a single experiment.(G) pERK1 and pERK2 pY/pT activation site phosphorylation kinetics following stimulation with EGF. Lines represent average of two technical replicates.