Protease and phosphatase inhibitors cocktail

Western blotting was performed as previously described [2] (link). Briefly, total proteins from HUVECs or thoracic aortic endothelial tissue were extracted using RIPA buffer supplemented with protease and phosphatase inhibitors cocktail (P1045, Beyotime Biotechnology). Protein concentration was determined based on the enhanced BCA protein assay kit (P0010, Beyotime Biotechnology), and stored at −80 °C for further analysis.
To evaluate protein expression level, proteins in 50 μg of the sample were separated using electrophoresis in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electro-transferred to polyvinylidene difluoride (PVDF) membranes and blocked for 1 h at room temperature using 5% non-fat milk. The membranes were incubated overnight at 4 °C with specific primary antibodies: anti GAPDH (1:1000 dilution), NMDA-R1 (1:1000 dilution), Sirt1 (1:1000 dilution), AMPKα (1:1000 dilution) and eNOS (1:1000 dilution). The membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:5000). Protein bands were visualized after incubating the membranes with an ECL reagent (E1050, Lablead). The intensity of the bands was measured using Image J software.