Time dependence of tyrosine phosphorylation of proteins from platelets activated by alboaggregin A in the presence of echicetin.

Washed platelets (700 μL) were stirred at 1100 rpm at 37°C with or without echicetin (15 μg/mL). After the addition of 0.3 μg/mL alboaggregin A, aliquots were removed at the times indicated and dissolved in SDS buffer containing inhibitors. The time point 00 represents the control before the addition of echicetin and alboaggregin A, whereas the time point 0 was after echicetin was added. After separation by SDS-PAGE (7%-17% acrylamide gradient) and transfer to PVDF membranes, the proteins were incubated with the antiphosphotyrosine Ab 4G10 before detection by using a peroxidase-linked second Ab and chemiluminescence.