2.12. Western blotting

Protein expression of different biomarkers related to thrombosis was evaluated in tissue (aorta) and cell (platelets and VSMCs) extracts by immunoblot. Following standard procedures, proteins (30-50 μg) were separated by SDS-PAGE under reducing conditions, transferred onto nitrocellulose membranes, incubated with primary and secondary-horseradish peroxidase conjugated antibodies, and revealed using the colorimetric kit Opti-4CN (BioRad, Hercules, CA, USA). Protein expression levels were normalized against β-actin and quantified using Image J software (available at https://imagej.nih.gov/ij/). The following antibodies and dilutions were used: Cyclooxygenase 2 (COX-2) – 1:500 (D5H5 #12282 – Cell Signaling Technology, Danvers, MA, USA); inducible nitric oxide synthase (iNOS) – 1:200 (M-19 #sc-650 Santa Cruz Biotechnology, Dallas, TX, USA); tissue factor (TF) – 1:500 (I-20 #sc-23596 – Santa Cruz Biotechnology, Dallas, TX, USA); kallikrein-1 (KLK1) – 1:500 (13G11 #19901 – QED Bioscience, San Diego, CA, USA); factor X (FX) – 1:500 (C-20 #sc-16341 – Santa Cruz Biotechnology, Dallas, TX, USA); factor II (FII) – 1:500 (D-15 #sc-23355 – Santa Cruz Biotechnology, Dallas, TX, USA); protease activated receptor – 1 (PAR1) – 1:500 (ATAP2 #sc-13503 – Santa Cruz Biotechnology, Dallas, TX, USA); plasminogen activator inhibitor – 1 (PAI-1) – 1:1000 (M-20 #sc-6644 – Santa Cruz Biotechnology, Dallas, TX, USA); urokinase plasminogen activator (uPA) – 1:500 (#CSB-PA14319A0Rb, Cusabio Biotech Co., Wuhan, China); and β-actin – 1:1000 (#A-1978, Sigma-Aldrich, Saint Loius, MO, USA).