Surface plasmon resonance measurements and quantitative Western blotting of the CLEC-2-Syk SH2 interaction. (A) Biotinylated CLEC-2 and FcRγ chain peptides were bound to streptavidin-coated biosensor chip surfaces. The N-SH2, C-SH2, or the tandem SH2 domains of Syk were purified and flowed over the chip at a range of concentrations. Nonlinear regression was used to analyze the data and calculate K_D values. The results are representative of 3 experiments. (B) GST-tagged Syk SH2 domain proteins were incubated with a 50-fold excess of biotinylated phospho-CLEC-2 peptide and precipitated with glutathione-agarose beads. The precipitated proteins were dot-blotted, and the amount of associated CLEC-2 was measured using HRP-streptavidin and densitometric analysis. The result is representative of 4 experiments.