Aortas were used as a surrogate large elastic artery to provide sufficient tissue for analysis of protein expression by western blot and enzyme activity as described previously (Cernadas et al. 1998 (link); Blackwell et al. 2004 (link); Ungvari et al. 2008 (link); Lesniewski et al. 2009 (link)). Aortas were excised, cleared of surrounding tissues and frozen in liquid nitrogen before storage at -80°C. For assay the tissue was pulverized over liquid nitrogen and homogenized in ice-cold RIPA lysis buffer containing protease and phosphatase inhibitors (Protease Inhibitor Cocktail Tablet (Roche) and 0.01% phoshatase inhibitor cocktail (Sigma)). Fifteen μg of protein was loaded on 12% polyacrylamide gels, separated by electrophoresis and transferred onto nitrocellulose membranes for western blot analysis. Antibodies for western analysis included anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling, Danvers, MA), anti-p67phox, anti-eNOS (BD Biosciences, San Jose, CA), anti-nitrotyrosine, anti-SIRT1, anti-catalase (Abcam, Cambridge, MA), anti-manganese superoxide dismutase (MnSOD) (Stressgen, Ann Arbor, MI). Enzyme activity of MnSOD was determined in aortic lysates (1 μg protein) using a superoxide dismutase (SOD) activity assay kit in the presence of 1 mmol/L potassium cyanide to block copper-zinc SOD (CuZnSOD) activities. Enzyme activity for catalase was measured using a kit (Cayman Chemical Ann Arbor, MI). NADPH oxidase activity (10 μg total protein) was measured using a Amplex red xanthine/xanthine oxidase assay kit (Invitrogen, Carlsbad, CA) according to manufacturer instructions with NADPH (200 μmol/L/reaction) as the reaction substrate. Pro-inflammatory cytokines interleukin-1 beta, interleukin-6, interferon gamma and tumor necrosis factors alpha were measured using a multiplex SearchLight Chemiluminescent Array Kit (Thermo Fisher Scientific Inc.) according to manufacturer's instructions.