Anti cox 1 antibody

Equal amounts of protein from gastric mucosal lysates were subjected to SDS-PAGE analysis, and immunoblotting using the appropriate antibodies was performed as we previously described.15 (link) The incubation condition for each antibody is as follows: anti-cPLA₂ antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-COX-1 antibody (1:1,000; Santa Cruz Biotechnology), anti-COX-2 antibody (1:1,000, Cayman Chemical), anti-HO-1 antibody (1:1,000; Abcam Inc., Cambridge, UK), anti-NQO-1 antibody (1:1,000; Abcam Inc.), anti-GCLC (1:1,000; Abcam Inc.), or anti-GCLM (1:1,000; Abcam Inc.).

Western blot analyses were performed as described previously.25 (link) Total protein was extracted by mammalian protein lysis buffer (Thermo Fisher Scientific). Equivalent amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with the following primary antibodies overnight at 4°C: anti-COX-2 antibody (Santa Cruz Biotechnology Inc, Dallas, TX, USA; 1:200 dilution), anti-COX-1 antibody (Santa Cruz Biotechnology Inc; 1:500 dilution), anti-hypoxia inducible factor-1 alpha (HIF-1α) antibody (Cell Signaling Technology, Inc, Danvers, MA, USA; 1:800 dilution), anti-vascular endothelial growth factor (VEGF) antibody (Santa Cruz Biotechnology Inc; 1:400 dilution), and anti-beta-actin (β-actin) antibody (Santa Cruz Biotechnology Inc; 1:1,000 dilution). The blots were detected by a secondary antibody coupled to horseradish peroxidase (HRP; Santa Cruz Biotechnology Inc) and an enhanced chemiluminescence system (EMD Millipore). The density of specific protein bands was determined by Image-Pro Plus software (v 5.0; Media Cybernetics, Inc, Rockville, MD, USA).