Platelet Protein Expression Analysis

Western blot analysis was performed as previously described by us (21 (link)). Platelet samples (−80°C) were thawed and tubes dissolved in 100 μL ice-cold PBS containing a protease inhibitor cocktail (P-8340; Sigma). Samples were then sonicated using an ultrasonic device, centrifuged at 14,000 × g for 10 min at 4°C; the extracts were denatured (10 min, 70°C), and 18 μg was loaded onto 10% bis–tris SDS-polyacrylamide gels (Thermo Fisher Scientific, Vienna, Austria), separated for 35 min at 200 V and finally electrotransferred to nylon-PVDF Immobilon-PSQ membranes for 20 min at 30 V in 20% methanol blotting buffer. Next, blots were blocked for 30 min in blocking buffer; incubated with primary antibody against APP (Abcam ab32136, 1:2,000, Cambridge, UK), or CD41 (Abcam ab63323, 1:2,000), or actin (1:1,000, A2066; Sigma, Vienna, Austria) at 4°C overnight; washed; and then incubated in alkaline phosphatase–conjugated anti–rabbit IgG for 30 min. After washing, bound antibodies were detected using an enhanced chemiluminescence system and visualized by using a cooled CCD camera (SearchLight; Thermo Fisher Scientific).