INU1 recognizes murine CLEC-2. (A) Flow cytometric detection of CLEC-2 on mouse platelets. Platelets were incubated with INU1-FITC (solid line) or an irrelevant rat IgG-FITC (shaded area) at saturating concentrations for 15 minutes at room temperature and analyzed directly. (B) Immunoprecipitation of CLEC-2 from surface-biotinylated mouse platelets. NP-40 lysates were incubated with 10 μg/mL INU1, followed by protein G–Sepharose. Proteins were separated on a 12% SDS-PAGE gel under reducing (red.) or nonreducing (n.r.) conditions, transferred onto a polyvinylidene difluoride membrane, and detected by streptavidin-HRP and ECL. (C) Binding of INU1 to mCLEC-2-Fc or mGPVI-Fc was tested by ELISA. The anti–mouse GPVI antibody, JAQ1, was used as a control (n = 3). Results are expressed as mean OD₄₅₀ nm ± SD. (D) Washed platelets from control mice were incubated with the indicated concentrations of INU1, and light transmission was recorded on a Fibrintimer 4-channel aggregometer. The results are representative of 4 individual experiments. (E) Washed platelets were stimulated with vehicle (rest.), 0.24 μg/mL rhodocytin (RC), 20 μg/mL INU1, or 1 μg/mL convulxin (CVX), lysed after 90 seconds, and probed with the phosphotyrosine-specific antibody 4G10 and ECL.