M per mammalian protein extraction reagent

The anticoagulated blood sample was centrifuged at 220×g for 20 min, and the platelet-rich plasma (PRP) was collected and transferred to a new tube for centrifugation at 480×g for 20 min at RT. The supernatant (plasma) was collected and stored at − 80 °C for further ELISA assay to detect the concentration of TXB2 (ADI-900-002, Enzo Life Sciences). Then, suspend the pellet (pure platelet) with 10 volumes of Thermo Scientific M-PER Mammalian Protein Extraction Reagent containing protease/phosphatase inhibitor Cocktail (Cat:5872, Cell Signal). The suspension was subjected to three cycles of sonication of 5 s each and centrifuged at 13,000×g at 4 °C for 10 min. 50 µg total protein per lane was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated in blocking buffer (5% w/v nonfat dry milk in Tris-buffered saline, 0.05% Tween-20 [TBS-T]) for 1 h at RT, and then incubated with primary antibodies overnight at 4 °C. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at RT for 1 h.