Anti nos3

Total protein extracts were obtained after washing the hearts with PBS and adding 300 ml of RIPA modified lysis buffer (50 mM NaCl, 50 mM Tris–HCl (pH 7.40), 1% Triton X-100, 1 mM EDTA, 1 mM PMSF; 2.5 g/l Protease Inhibitor Cocktail (Sigma-Aldrich Co., St. Louis, MO, USA), 1 mM Na₃VO₄, 1 mM NaF), or washing the cultured cells and scraped off the dishes with 50 µl of the same buffer. Then, the tubes were kept on ice for 30 min with swirling, and the samples were centrifuged at 7,000 g at 4°C for 10 min. The supernatants were stored at −20°C. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad, USA) and bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) as a standard (36 (link)). For Western blot analysis, total proteins were boiled in Laemmli sample buffer, and equal amounts of protein (40–50 µg) were separated by 10–12% SDS-PAGE. The gels were blotted onto a Hybond-P membrane (GE Healthcare, Madrid, Spain) and incubated with the following antibodies: anti-NOS2, anti-NOS3, anti-Arginase I (Arg-I), anti-CD31, anti-VEGF-A, and anti-α-actin (Santa Cruz Biotechnology, CA, USA). The blots were revealed by enhanced chemiluminescence in an Image Quant 300 cabinet (GE Healthcare Biosciences, PA, USA) following the manufacturer’s instructions. Band intensity was analyzed using the NIH-ImageJ software (37 (link)).