Increased tyrosine phosphorylation of Btk and Tec in collagen-stimulated platelets.

(A) Aliquots of platelets were treated with 50 μg/mL of either collagen or buffer for 5 minutes, then were lysed in detergent buffer as described in “Materials and methods.” Btk or Tec was purified from the soluble extracts by immunoprecipitation, and the denatured samples were divided into 3 equal aliquots. Replicate pairs of the samples (without or with collagen treatment) were separated by 7.5%-15% SDS-PAGE, transferred to a nitrocellulose membrane, then immunoblotted with either 4G10 total phosphotyrosine antibody (top panel), anti-Btk antibody (middle panel), or anti-Tec antibody (bottom panel). (B) Platelets were treated with 50 μg/mL of either collagen or buffer for 10 minutes or less. Btk was purified from the soluble extracts by immunoprecipitation, as described above, then immunoblotted with either 4G10 total phosphotyrosine antibody (top panel) or anti-Btk antibody (bottom panel). (C) Platelets were incubated in nominally Ca⁺⁺– and Mg²⁺–free modified Hepes-Tyrode buffer and treated with 50 μg/mL of either collagen or buffer for 5 minutes, followed by the addition of 10 mmol/L EDTA. Btk was purified from the soluble extracts by immunoprecipitation as described above, then immunoblotted with either 4G10 total phosphotyrosine antibody (top panel) or anti-Btk antibody (bottom panel). (D) Aliquots of platelets, as described in (C), were treated with 50 μg/mL of either collagen or buffer for 0 to 10 minutes following the addition of 10 mmol/L EDTA. Tec was purified from the soluble extracts by immunoprecipitation as described above and immunoblotted with either 4G10 total phosphotyrosine antibody (top panel) or anti-Tec antibody (bottom panel).