YxxL is essential for Syk association and signaling through CLEC-2. (A) Washed platelets (5 × 108/mL) were lysed with 2 times NP40 lysis buffer, precleared, and interacting proteins precipitated with the addition of 10 μg of the relevant biotinylated CLEC-2 peptide. Precipitated proteins were separated by SDS-PAGE and Western blotted for the presence of Syk. (B) DT40 cells were transfected with 10 μg/mL of the stated CLEC-2 construct and an NFAT-luciferase reporter plasmid. Transfected cells were stimulated with 50nM rhodocytin for 6 hours at 37°C, and then the luciferase activity was measured as an index of signaling. Results were normalized for transfection efficiency and plotted as a percentage of the wild-type response. Error bars represent the geometric mean ± SE of 3 to 6 separate experiments.
