AYP1 recognizes CLEC-2 on human platelets. (A) Washed platelets were incubated for 5 minutes at 37°C under stirring conditions in the absence and presence of 100 nM rhodocytin, followed by lysis and immunoprecipitation with 2 µg/mL of goat α-human CLEC-2 (previously characterized antibody), AYP1, or IgG1 coupled to protein-G sepharose. Proteins were separated on a 12% SDS-PAGE gel under reducing conditions, transferred onto a polyvinylidene difluoride membrane, and probed with AYP2. Membranes were subsequently stripped and reprobed with α-phosphotyrosine antibody 4G10. (B) Deglycosylation reduces CLEC-2 to a single band. Lysates of resting platelets were incubated in the absence or presence of peptide-N-glycosidase F (PNGase F), probed with AYP2, and stripped and reprobed with an antibody against α-tubulin. (C) Analysis of AYP1 binding to 293T Rex cells with doxycycline-inducible protein expression of CLEC-2. Cells were incubated in the absence or presence of 1 µg/mL of doxycycline for 24 hours, and AYP1 binding was determined by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated sheep α-mouse secondary antibody. (D) Flow cytometric analysis of CLEC-2 on platelets and leukocytes. Platelets and leukocytes were isolated and incubated with saturating concentrations of either Alexa Fluor-488 (AF-488) conjugated α-CLEC-2 antibody AYP1 or isotype-matched control for 30 minutes at room temperature (platelets) or on ice (leukocytes) and analyzed immediately. Leukocyte subset discrimination is described in the supplemental Methods and the gating strategy is shown in supplemental Figure 1A. Data are representative of ≥3 independent experiments.