4.4. SDS-PAGE and Western Blot Analysis

Platelet samples for Western blot analysis were prepared by adding 3× Lämmli buffer (200 mM tris/HCl, 15% (v/v) glycerol, 6% (w/v) SDS, 0.06% (w/v) bromphenol blue, 1:10 β-mercaptoethanol) directly in the aggregation cuvettes to stop platelet responses, then boiled at 95 °C for 10 min under gentle shaking. Platelet proteins were separated by electrophoresis using 8% SDS-polyacrylamide gels followed by immunoblotting as previously described [28].

Phospho-antibodies against Syk S297, Syk Y525/526, Syk Y352, LAT Y191, PLCγ2 Y759, and MARCKS S159/163 (Cell Signaling Technologies, Danvers, MA, USA) were used diluted 1:1000 in 5% BSA or 1:700 for MARCKS. Blots probed with phospho-antibodies were stripped and reprobed with a corresponding antibody detecting the total protein, anti-Syk, or anti-PLCγ2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or other loading controls anti-β-actin or anti-α-actinin (Cell Signaling Technologies, Danvers, MA, USA) diluted 1:1000 in 5% BSA. After incubation with the appropriate secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (BioRad Laboratories, Hercules, CA, USA) enhanced chemiluminescence (ECL) detection was performed using Fusion FX7 (Vilber Loumat GmbH, Eberhardzell, Germany).