CLEC-2 oligomers are present on the platelet surface. Washed platelets (5 × 108/mL) under basal or rhodocytin-stimulated (100nM) conditions had their surface proteins cross-linked with the addition of 0.15mM or 1.5mM Sulfo-EGS cross-linking reagent, with a linker length of 1.6 nm (16 Å). The cross-linking reaction was subsequently blocked, and then the platelets were lysed with 2× NP40 lysis buffer. (A) Lysates were precleared with protein G-Sepharose and then immunoprecipitated with α-CLEC-2 antibody and protein G-Sepharose. Precipitated proteins were separated by reducing SDS-PAGE and Western blotted for CLEC-2. (B) Lysates were separated by reducing SDS-PAGE and Western blotted for CLEC-2, FcγRIIA, and Src. The results are representative of 3 experiments.
