Glyceraldehyde 3 phosphate dehydrogenase gapdh

Protein concentrations were determined using a BCA protein assay kit. Equal amounts (20 μg/lane) of protein were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 70–120 V. The proteins were next transferred to polyvinylidene difluoride (PVDF) membranes through electroblotting for 60 min at 100 V. The transferred membranes were then blocked with 5% non-fat milk [1 g non-fat milk dissolved in 20 mL Tris-buffered saline with 0.05% Tween-20 (TBST)] for 2 h on a shaking table. Subsequently, the membranes were incubated with primary antibodies, including anti-eNOS (1:1000; Abcam, MA, USA), p-eNOS Ser 1177 (1:1000; Abcam, MA, USA), RAGEs (1:1000; Abcam, MA, USA), sirtuin-3 (SIRT3; 1:1000; SantaCruz, Texas, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; GeneTex, CA, USA) at 4 °C overnight on a shaking table. The membranes were then probed with their respective secondary antibodies conjugated with horseradish peroxidase including rabbit IgG antibody and mouse IgG antibody (1:10000; GeneTex, CA, USA) at room temperature for 50 min. After the probing process, each of the membranes was washed three times with TBST for 5 min. The bands were then visualized using an enhanced chemiluminescence detection kit (ECL kit) (Millipore, Merck KGaA, Darmstadt, Germany). The protein bands were quantified using Image J software.