Imagequant las 4000 mini densitometer

COX-1, COX-2, EP1–EP4 receptors and mPGES-1 protein expression from homogenate cells were detected by Western blotting. Briefly, MT-III-stimulated and non-stimulated cells were lysed with 100 mL of a sample buffer (0.5 M Tris-HCl, pH 6.8, 20% SDS, 1% glycerol, 1 M β-mercaptoethanol, 0.1% bromophenol blue) and boiled for 10 min. Samples were resolved by SDS-PAGE on 10% bis-acrylamide gels overlaid with a 5% stacking gel. Proteins were then transferred to nitrocellulose membranes using a Mini Trans-Blot (Bio-Rad Laboratories, Richmond, CA, USA). Membranes were blocked for 1 h with 5% albumin in Tris-buffered saline (20 mM Tris, 100 mM NaCl and 0.5% Tween 20, pH 7.2) and incubated overnight with primary antibodies against COX-1 and COX-2; EP1, EP2, EP3, and EP4 receptors; and mPGES-1 (1:500 dilution) or β-actin (1:3000 dilution) for 1 h at room temperature. Membranes were then washed and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. Immunoreactive bands were detected by the entry-level peroxidase substrate for enhanced chemiluminescence, according to the instructions of the manufacturer (GE Healthcare). Band densities were quantified with an ImageQuant LAS 4000 mini densitometer (GE Healthcare) using the image analysis software ImageQuant TL (GE Healthcare).

Western blot analysis was implemented as described previously (Ma et al., 2019 (link)). In brief, aliquots of total protein extract (20 μg) from cells were loaded and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes (PVDF; EMD Millipore, Billerica, MA, United States). Blots were blocked with 5% non-fat milk in PBST (PBS containing 0.1% TWEEN-20) for 1 h at room temperature, and the membrane was incubated with primary antibody against PPARG (Abcam, ab178860) at 4°C overnight. The membrane was then washed three times with PBST and exposed to horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2000 dilution, Cell Signaling Technology, United States) for 1 h at room temperature. After three washes with PBST, antibody binding was detected with enhanced chemiluminescence substrates (Millipore, United States) and visualized with an ImageQuant LAS 4000 mini-densitometer (GE Healthcare Life Sciences, United States).