Laminin-induced protein phosphorylation is dependent on GPVI. Washed murine platelets (4 × 10⁸/mL) from control or GPVI-deficient mice (GPVI^-/-) were pretreated with 1 mM GRGDS peptide prior to adhesion to laminin as described in Figure 1. BSA-coated dishes were used for negative control; 300 μL of washed platelets were seeded on dishes for the indicated times at room temperature prior to stopping the reaction by addition of 300 μL of 2 × lysis buffer. (A) Proteins were separated by 6% to 20% gradient SDS-PAGE and protein-tyrosine phosphorylation visualized by Western blotting with 4G10. Protein loading was measured by Western blotting with anti-PLCγ2 pAb. (B) Fc receptor γ chain (i), SLP-76 (ii), LAT (iii), Btk (iv), Syk (v), or PLCγ2 (vi) were isolated by immunoprecipitation using specific antibodies before blotting with 4G10. Gels were reprobed with the antibody that was used in the immunoprecipitation studies. Tyrosine phosphorylation of Syk and PLCγ2 were quantified using Quantity One software for Macintosh. Optical density measurements were standardized by the recruitments of these proteins. The optical densities are shown above the corresponding lanes. The results are representative of 2 experiments.