2.7. Western Blot

Cell lysates were run on a 4–12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel (Bio-Rad, Hercules, CA, USA) and the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk in 1% TBST (Tween-20 tris-buffered saline) for 1 h at RT and then incubated with a primary antibody specific for iNOS (ab129372; Abcam) overnight at 4 °C. The membrane was washed and then incubated with the appropriate horseradish peroxidase secondary antibody for 1 h at RT. The protein blots were incubated with ECL substrates (Bio-Rad) for 5 min at RT and then detected on CL-Xposure Film (Thermo Scientific, Skokie, IL, USA) and developed.