Western Blot Analysis of COX-2 and PGE2

Western blots were performed as described in our previous studies [14 (link),36 (link)]. Specific primary antibody anti-COX-2 (1:600, Santa Cruz Biotechnology) or anti-PGE2 (1:700; Bioss Antibodies) was mixed in 1× PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20, and incubated at 4 °C, overnight. After that, blots were incubated with the peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with the antibody against beta actin (1:1000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003).