Association of CLEC-2 with rhodocytin-coated beads. (A) Washed platelets were labeled with biotin and lysed with an equal volume of 2 × lysis buffer. They were precleared and incubated with rhodocytin-bound (rhod-Seph) or glycin-bound (gly-Seph) Sepharose 4B for 4 hours at 4°C. After extensive washing, proteins were eluted from the beads with SDS-reducing sample buffer. Precipitated platelet proteins were separated by 4% to 20% SDS-PAGE and detected by horseradish peroxidase–conjugated streptavidin (avidin-HRP). Data are representative of 2 experiments. (B) Pull-down and electrophoresis were performed as described in panel A using unlabeled washed platelets. Ligand blotting was performed by biotin-conjugated rhodocytin (rhodocytin-biotin) and avidin-HRP. Arrows indicate 32-kDa protein that was precipitated specifically by the rhodocytin-coupled beads. (C) Washed human platelets were incubated with control goat IgG or anti–CLEC-2 antibody, followed by staining with FITC-conjugated anti-goat IgG. Samples were analyzed with a Becton Dickinson FACScan. (D) Washed platelets were dissolved in 4 × SDS sample buffer, separated by SDS-PAGE, and blotted with anti–CLEC-2 antibody (left) or control goat IgG (right). The data are representative of 5 experiments. (E) The membrane used in panel B was reprobed with anti–CLEC-2 antibody. (F) Densitometric analysis of 32- and 40-kDa CLEC-2 bands in panel D was performed by Molecular Imager FX and Quantity One software (Bio-Rad Laboratories, Hercules, CA). Relative intensity of the 40-kDa band compared with the 32-kDa band was expressed as mean ± SE (n = 4). (G) CLEC-2 immunoprecipitates were incubated with 1.5% 2-mercaptoethanol for 3 minutes at 100°C. After addition of 1.4% Nonidet P-40, the proteins were incubated with or without N-glycosidase F at 37°C overnight. Immunoprecipitated and enzyme-treated CLEC-2 protein was dissolved with SDS sample buffer, separated by SDS-PAGE, electrotransferred, and Western blotted by the CLEC-2 antibody.