Anti timp 2

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.