Immunoblotting Quantification of Washed Platelets

Washed platelets (4 × 10⁸/mL) were stimulated as indicated and lysed directly in 4X NuPAGE sample buffer containing 0.5 M dithiothreitol (DTT). Proteins were resolved by electrophoresis as previously described^{45 (link)} and transferred onto polyvinylidene fluoride (PVDF) membranes. For some experiments, gels and membranes were cut into two parts to be probed separately with different primary antibodies^{47 (link)}. Proteins were visualized by near-infrared detection using a LI-COR® Odyssey imaging system (providing a wide linear dynamic range) unless indicated otherwise. LI-COR® Image Studio (LI-COR®, Cambridge, UK) was used to create final images, the software analysis options only affect how raw data pixels are mapped to the screen and does not alter the experimental data. LI-COR® Image Studio (LI-COR®, Cambridge, UK) was used to quantify bands. Bands were defined using the rectangle shape tool to obtain fluorescence values and median local background (intensity of pixels in a border around the shape) was automatically subtracted.