Anti mmp 2

Inflamed and non-inflamed biopsies, obtained from the colon of IBD patients, were frozen, homogenized and then lysed using a lysis buffer composed of Tris-HCl 10 mM pH 7.4, EDTA 100 mM, NaCl 100 mM, SDS 0.1%, and protease inhibitor cocktail 1% (Sigma, Saint Louis, MO, USA). The protein concentration was determined using the Bradford reagent (Sigma, Saint Louis, MO, USA). Samples were run in a denaturing 10% polyacrylamide gel (Thermo Fisher Scientific, Carlsbad, CA, USA) and were transferred to a PVDF membrane (Thermo Fisher Scientific, Carlsbad, CA, USA) that was incubated overnight at 4 °C with primary antibodies (anti-actin 1:10000 (42 kDa), anti-MMP9 1:1000 (92 kDa), and anti-MMP2 1:1000 (74 kDa); Sigma Saint Louis, MO, USA). The secondary antibodies were an anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (Cell Signaling, Danvers, MA, USA), diluted 1:40000 and an anti-rabbit HRP-conjugated secondary antibody (OriGene, Herford, Germany) diluted 1:1000. The reaction was developed with a chemiluminescence reagent containing luminol (Euroclone, Milan, Italy). Chemiluminescence was developed using LiteAblot^® TURBO (Euroclone, Milan, Italy) and exposed on Kodak Biomax film. MMP protein expression was quantified using the ImageJ software, version 1.45s and was reported as percentage with respect to actin.

Liver samples were homogenized with lysis buffer (Cell Signal Technology Inc., Danvers, MA) and centrifuged at 20,000 ×g for 60 minutes at 4°C. The resultant supernatants were used as the total liver protein and subjected to western blotting. The protein concentrations were determined by Bradford's method. Proteins were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinyl difluoride membrane. Each blot was treated with the anti-b-actin antibody (β-actin 1 : 5,000; mouse monoclonal antibody; Sigma-Aldrich, St. Louis, MO), anti-matrix metalloproteinase-13 (MMP-13, 1 : 400), antitissue inhibitors of metalloproteinase-1 (TIMP-1, 1 : 400), anti-MMP-2 (1 : 300), and anti-TIMP-2 (1 : 400). All the above were from Santa Cruz Biotechnology. Then, secondary antibody (Santa Cruz Biotechnology) was added, and the reaction bands of western blot were quantified by Quantity One software (Bio-Rad Laboratories, Inc., Berkeley, CA) and modified by the β-actin. The values (% of control) are given as means ± SD of 5 animals.

Whole-cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore) using a semidry transfer system (Bio-Rad, Hercules, CA, USA). The membranes were probed with specific antibodies and then incubated with horseradish peroxidase-conjugated antibodies against mouse or rabbit immunoglobulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by detection with enhanced chemiluminescence western blotting detection reagent (GE Healthcare, IL USA). An ImageQuant LAS4000 mini system (GE Healthcare) was used as a chemiluminescence detector. The following antibodies were used in this study: anti-TIMP-2 (1:1000; cat. no. SAB1400279; Sigma-Aldrich, St. Louis, MO, USA), anti-MMP-2 (1:2000; cat. no. 13132; Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (polyclonal; 1:50000; cat. no. A5316; Sigma-Aldrich). Densitometric analysis was performed using NIH Image J software.