Anti il 17

Aortic tissues were homogenized in cold lysis buffer (10 mM Tris (pH 7.5), 1 mM sodium orthovanadate, 1% SDS). Protein concentration was measured by the bicinchoninic acid method (Bio-Rad, Hercules, CA, USA). Homogenate samples were run on 10% SDS-polyacrylamide gels and stained with Coomassie blue. Gel images were scanned and quantified as described below, to adjust for equal loading. After correction of protein concentrations, equal amounts of protein were run on 10% SDS-polyacrylamide gels and electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The blots were blocked with 5% non-fat milk in PBS-T (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, 0.1% Tween 20 pH 7.5) for 30 min at room temperature and then incubated overnight at 4 °C with primary antibody (anti-ICAM-1 (Cell Signaling Technology, Boston, MA, USA) or anti-IL-17 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the blots were incubated at room temperature for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology) diluted 1:5000 in blocking solution. Antibody binding was visualized using the enhanced chemiluminescence system (Amersham Bioscience, Buckinghamshire, UK). Specific bands were scanned and quantified using the Quantity One software (Bio-Rad).