Western blotting for native eNOS and phospho‐eNOS(Ser1177)

Protein expression of endothelial NOS (eNOS) and phospho‐eNOS(Ser¹¹⁷⁷) was performed in non‐denaturating conditions as previously described (Yamamoto et al., 2007). Briefly, rat hearts were lysed in ice‐cold protein lysis buffer under native conditions (50 mM Tris‐HCl, pH 7.4; 150 mM NaCl; 5 mM CaCl₂; protease and phosphatase inhibitor cocktails). Protein concentration was determined, and 20 μg of native total protein was diluted in 5×non‐denaturating loading buffer (250 mM Tris‐HCl, pH 6.8; 50% glycerol; 0.5% w/v bromophenol blue). Both electrophoresis and blotting procedures were performed at 4°C. Samples were separated by SDS‐PAGE on 10% bis‐acrylamide gels in SDS‐free buffer and transferred onto PVDF membranes. Membranes were probed with anti‐eNOS and anti‐phospho‐eNOS(Ser¹¹⁷⁷) antibodies.