Coprecipitation of GPVI and calmodulin from platelet lysates.

(A) Immunoprecipitation (IP) by anti-GPVI antibody (MM20411) of untreated platelet lysates or lysates of platelets (5 × 10⁸/mL) treated with 10 μg/mL collagen-related peptide. Samples were electrophoresed on 10% or 15% polyacrylamide gels under reducing conditions and were immunoblotted with anticalmodulin (Anti-CaM) IgG. The position of the antibody light chain is shown. Blots were visualized using horseradish peroxidase–coupled second antibody (Silenus, Hawthorn, Australia) and enhanced chemiluminescence detection (ECL; Amersham, Buckinghamshire, United Kingdom). Samples were also ligand blotted with convulxin (Cvx; lower panel) to determine GPVI levels.12 WB indicates Western blot. (B) Immunoprecipitation by anti-GPVI IgG of untreated platelet lysates or lysates of platelets treated with 10 μg/mL collagen (coll) or collagen-related peptide (CRP), or 200 nM A23187 for 90 seconds (upper panel) or 30 seconds (lower panel) at 37°C. Where indicated, platelets were preincubated with 20 μM PP1 before stimulation with collagen-related peptide. Samples were immunoblotted with anticalmodulin IgG as described above. Experiments with collagen-related peptide were performed 6 times, and those with collagen were performed 3 times. (C) Coprecipitation of calmodulin with amylose–Sepharose beads from platelet lysates in the presence of MBP or MBP-GPVI (Glu266-Ser316) fusion protein. Precipitates were electrophoresed on 5% to 20% sodium dodecyl sulfate–polyacrylamide gels under reducing conditions, electrotransferred to nitrocellulose, probed with anticalmodulin IgG or anti-MBP IgG, and visualized using the ECL reagent. Results are typical of 3 separate experiments.