Image studio software lite ver 5

Catalase activity in colonic mucosa was assessed colorimetrically in a reaction with 0.03% H₂O₂ solution. The reaction was stopped by the molybdate ammonium (Alfarus, Ukraine) and measurement was taken at a wavelength of 410 nm. The activity of superoxide dismutase (SOD) was determined by in-gel activity assay [37 (link)–38 (link)]. Colonic mucosa samples were electrophoresed in 10% native-polyacrylamide gel at 40 mA at 4°C. After electrophoretic separation, the gel was incubated for 20 min in 50 mM phosphate buffer solution (pH 7.8) containing 0.25 mM NBT, 1 mM EDTA, 28 mM TEMED, and 0.028 mM of riboflavin (Sigma-Aldrich, Germany). Gel was developed by illumination. Achromatic bands on the light-blue background of the gel indicated the presence of SOD. Densitometry was performed using LI-COR Image Studio Software Lite Ver. 5.2.

Total protein cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA buffer, 1% NP-40, 50 mM Tris pH 7.6, 120 mM NaCl, 1 mM EDTA) with protease and phosphatase inhibitors (Sigma-Aldrich P8340 and P0044), quantified using the Bicinchoninic acid protein assay method and analysed by SDS-PAGE as described previously [1 (link)]. Western blotting was performed with primary antibodies to PPARγ (C26H12), C/EBPα (2295), β−actin (8H10D10), active β-catenin (D13A1) (Cell Signalling Technologies ^®, Leiden, The Netherlands), aP-2 (C-15), collagen type III alpha 1 (B-10 (Santa Cruz Biotechnology Inc) or collagen III (ab7778, Abcam PLC, Cambridge, UK) at 1:1000 dilution. IRDye® secondary antibodies (LI-COR™, Lincoln, NE, USA) for detection with an Odyssey FC image analyser (LI-COR®) was used and images were analysed with Image Studio Software Lite Ver 5.2 (LI-COR^®). Quantitation was relative to nitrocellulose-bound total protein, stained using REVERT™ total protein staining kit (926-1101, LI-COR™) and analysed at 700 nm with the Odyssey FC image analyser, unless otherwise stated.