Western blot analysis and eNOS dimer blot analysis

Cells were lysed in SDS-sample buffer (62.5 mMTris, pH 6.8, 2% SDS, and 10% glycerol) and then sonicated for 5 s to reduce sample viscosity. Each sample was resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA, USA), analyzed with antibodies [eNOS, inducible nitric oxide synthase (iNOS), hArg1, hArg2 antibodies from Santa Cruz Biotechnology and phospho-eNOS Ser1177 and phosphoeNOS Thr495 antibodies from BD Biosciences] according to the suppliers' protocols, and visualized with peroxidase and an enhanced-chemiluminescence system (Thermo Scientific Pierce, Waltham, MA, USA). Normalization was performed using an anti-β-tubulin antibody (BD bioscience, 1:1000). A densitometry analysis of bands was performed using NIH ImageJ. Dimers and monomers of eNOS were separated by low-temperature SDS-PAGE and analyzed as described above.