Mouse monoclonal anti cox 2

For protein preparation, Caco-2 cells were lysed in PRO-PREP^TM buffer (iNtRON Biotechnology, Kirkland, WA, USA). For the immunoblot assay, supernatants containing 40 µg of protein were loaded into individual lanes of an 8% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and immunoblotted onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). The membrane was then incubated with mouse monoclonal anti-COX-2 (1:1,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) or rabbit polyclonal anti-iNOS (1:1,000 dilution; Abcam) for 2 h. Bound antibodies were detected using a chemiluminescent substrate (Miracle-Star^TM; iNtRON Biotech, Gyeonggi, Korea), according to the manufacturer’s instructions. After imaging, the membranes were stripped and reprobed with mouse monoclonal anti-β-actin (1:10,000 dilution; Sigma-Aldrich). The optical density (OD; per mm²) of each band was measured, and the density of the band relative to the density of that of β-actin was compared using ImageJ software (NIH, Bethesda, MD, USA).

Cell pellets obtained from each experiment were mechanically homogenised in lysis buffer (RIPA supplemented with protease inhibitors cocktail) and total amount of protein was determined by using Bradford assay. Denatured proteins (60 µg) were separated on 10% sodium-dodecylsulfate polyacrylamide gels and transferred to polyvinylidene fluoride membrane (PVDF). Membranes were blocked in phosphate-buffered saline containing 0.1% v/v Tween-20 (PBST) and 3% w/v non-fat dry milk for 30 min, followed by overnight incubation at 4 °C with primary antibodies: mouse monoclonal anti-iNOS (1:500, BD-Pharmaingen), mouse monoclonal anti-COX-2 (1:2000, SantaCruz Biotechnologies), mouse monoclonal anti-CSE (1:1000, Proteintech), rabbit polyclonal anti-CBS (1:1000; Santa Cruz Biotechnologies), rabbit polyclonal anti-3MST (1:500, Novus Biologicals)⁵⁰ or mouse monoclonal anti-tubulin (1:5000, Sigma-Aldrich). Membranes were extensively washed in PBST prior to incubation with horseradish-peroxidase conjugated secondary antibody for 2 h at room temperature. Following incubation, membranes were washed and chemiluminescence was detected by using ImageQuant-400 (GE-Healthcare). The target protein band intensity was normalised against housekeeping protein α-tubulin.