Thrombin-evoked rapid Btk phosphorylation and activation in human platelets. Platelets were rapidly mixed with thrombin at a final concentration of 0.1 U/mL, or with agonist-free HBS solution (control at 0 seconds) and incubated at 37°C for various time periods (0.1-2.5 seconds) before mixing with lysis buffer using a quenched-flow system. Proteins were separated by SDS-PAGE followed by Western blotting with either anti-phospho-Btk (Y-223) antibody (Ai) or anti-Btk antibody (Aii) as described in “Materials and methods.” Bands were revealed using chemiluminescence and were quantified using scanning densitometry. Positions of molecular-mass markers are shown on the right. (B) Graph represents the quantification of Btk phosphorylation. Values are mean ± SE of 6 runs made on cell preparations from 6 donors expressed as the percentage of Btk phosphorylation in resting cells.