Western lightning ultra chemiluminescence kit

Following aggregations, platelets were lysed by adding 62.5 μL of 4X SDS in 250 μL of washed platelets, and heated at 95°C for 5 minutes. Samples were stored at −20°C until further analysis. Proteins from the 3 mouse groups’ platelets were separated by 10% SDS‐PAGE followed by its transfer to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk for 1 hour at room temperature. Antibodies against CLEC‐2 (R&D Systems, AF1718) and pAkt (Cell Signalling, 9275) were incubated with the membranes overnight at 4°C, and a horseradish peroxidase–conjugated secondary antibody (Abcam, AB6741) was used for detection using the Western Lightning Ultra chemiluminescence kit (PerkinElmer).

Proteins were extracted using radioimmunoprecipitation assay buffer and the protein concentration was established using the Bradford Protein Assay Kit (Bio Basic). Protein samples were diluted in 4X Laemmli buffer, then heated at 95°C for 5 minutes. Proteins were separated by electrophoresis on a 12% SDS‐PAGE, then transferred on nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris‐buffered saline (TBST, 0.1% Tween 20) for 1 hour at room temperature, then incubated with an anti‐podoplanin (OriGene, DP3512S), an anti‐vascular endothelial growth factor receptor (VEGFR)‐3 (Abcam, AB27278), or an anti‐beta‐actin (Abcam, AB8227) overnight at 4°C. The membranes were washed with TBST and incubated with horseradish peroxidase–conjugated secondary antibodies (Abcam, AB6721 and AB6721) for 1 hour at room temperature. Western Lightning Ultra chemiluminescence kit (PerkinElmer) was used for detection. Each sample was normalized with its respective beta‐actin expression.