P enos ser1177

Protein expression was determined by Western blot analysis in homogenates of aortic tissue or MLECs. Separation of the proteins was performed by SDS-PAGE electrophoresis followed by immunoblotting onto nitrocellulose membranes [36 (link)]. Antibodies—α1AMPK (1:500; 07-350, Merck KGaA, Darmstadt, Germany), α-actinin (1:2000; 2044, Sigma-Aldrich, Germany), ß-actin (1:2000; A5060, Sigma-Aldrich, Germany), eNOS (1:1000; 610297, BD, Heidelberg, Germany), p-eNOS Ser1177 (1:1000; 612393, BD, Heidelberg, Germany), 3-nitrotyrosin (1:200; 05-233, Merck KGaA, Darmstadt, Germany) and Nrf-2 (1:200; sc13032, Santa Cruz, Dallas, TX, USA) were used according to the manufacturer’s protocols. The positive bands were detected using species-matched secondary antibodies with covalently bound peroxidase (1:10,000; GAM-POX: PI-2000 and GAR-POX—PI-1000, Vector Laboratories, Burlingame, CA, USA) and an enhanced chemiluminescence detection kit—Pierce™ ECL Western Blotting Substrate kit (32106, Thermo Scientific), and quantification by a Chemilux chemiluminescence imager (CsX-1400M, Intas, Göttingen, Germany).

Cells lysates (20–40 μg protein per sample) were separated under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA) and transferred onto a polyvinylidene fluoride membrane (PerkinElmer, Waltham, MA) by semidry electroblotting (36 (link)). Membranes were probed with antibodies against human eNOS (BD Bioscience, San Jose, CA; Abcam, Cambridge, MA; Cell Signaling Technology, Danvers, MA) and phospho-eNOS (P-eNOS Ser-1177; BD Bioscience), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SP1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), A20 (Abcam), and β-galactosidase (Novus Biological, Centennial, CO), followed by the appropriate secondary horseradish peroxidase-conjugated antibodies (Thermo Scientific, Rockford, IL). Protein bands were detected with enhanced chemiluminescence kit (PerkinElmer, Waltham, MA) after exposure to an autoradiography film. The intensity of the scanned bands was quantified by densitometry using the ImageJ 1.41 software (NIH, Bethesda, MD). Alternatively, IRDye® infrared secondary antibodies were used and WB imaging was digitally acquired using the Odyssey® CLx imaging System. The intensity of the bands was quantified using the Image Studio^TM Software (Li-COR Inc, Lincoln, NE).