Goat anti rabbit conjugated with horseradish peroxidase

Laemmli sample buffer (×4) (Bio-Rad) was added to 30 μg of each protein sample and the mixtures were boiled at 95 °C for 5 minutes. Proteins were then loaded on 7.5% SDS-PAGE gel (Bio-Rad) followed by transfer to nitrocellulose membrane for 1 hour at 100 V, 4˚C. The membrane was then blocked with 5% milk in TBST for 1 hour and probed with rabbit anti-α-tubulin (0.5 μg/ml) (Abcam) overnight at 4 °C. The membrane was further probed with goat anti-rabbit conjugated with horseradish peroxidase (1:10,000) (Jackson immunoresearch) for 1 hour. Bands were detected using chemiluminescence detection kit (Biological Industries), and quantification was performed by densitometry. The percentage change in depolymerized tubulin was determined by the following equation: (gained densitometric values of depolymerized tubulin/sum of gained densitometric values of polymerized and depolymerized tubulin) × 100.

Protein (15 μg) extracts from the platelets were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 2% non-fat dry milk for 1 hour and incubated with anti-VASP^Ser239 (Cat. no. 3114), anti-VASP^Ser157(Cat. no. 3111), anti-VASP (Cat. no. 3112) and GAPDH (Cat. no. 2118) overnight (Cell Signaling Technology, Danvers MA). Goat anti-rabbit conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) was used as a secondary antibody and followed by ECL detection (Bio-Rad, Hercules, CA). Band density was qualified by NIH ImageJ software.