3.7. Western Blot Quantification of COX-2 and iNOS Expression

Western blot quantification was measured to analyze COX-2 (abcam, Cambridge, MA, USA) and iNOS (sigma, St. louis, MO, USA) expression. Kidney tissues isolated from each animal were homogenized in KCL buffer with protease and phosphatase inhibitors. The cell lysates were centrifuged at 13,000 rpm for 30 min. The supernatant containing cytoplasmic fractions was isolated, and protein concentration was measured as described below. The cytoplasmic fraction containing equal amounts of protein (35 μg) were loaded and separated using 10% SDS–polyacrylamide gel electrophoresis. The proteins were then transferred onto a PVDF membrane using Trans-Blot Turbo Transfer System, (Bio-rad, Hercules, CA, USA). The membranes were washed and blocked using 5% skimmed milk. Next, the membranes were incubated overnight at 4 °C with specific primary rabbit polyclonal antibodies against COX-2 (1:1000) and iNOS (1:5000). The next day, the membranes were washed and then incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody. The protein recognized by the antibody was visualized using an enhanced chemiluminescence Pico kit (Thermo Fisher Scientific, Rockford, IL, USA). The blots were stripped and re-probed for β-actin (1:5000; monoclonal mouse, Millipore, St. louis, MA, USA) as a loading control. The intensity of the bands was measured using densitometry and quantified using Image J software (NIH, Bethesda, Rockville, MD, USA). Not applicable, download form google