Mmp 13

Antibodies used were phospho-PKCδ (Ser645), MMP-13, SOX-9, Collagen Type-X (Millipore, Billerica, MA), ADAMTS-5, (Thermo Fisher Scientific, Rockford, IL), RUNX-2, TIMP-3 (Abcam, Cambridge, MA), phospho-ERK1/2(Thr202/Tyr204), phospho-NF-κB-p65(Ser536) (Cell Signaling Technology, Danvers, MA).

Human articular chondrocyte cultures were extracted using RIPA lysis buffer containing protease inhibitors (Roche). Protein was extracted from mouse whole joints following removal of the skin and muscle bulk. Tissue was snap-frozen and then extracted using RIPA lysis buffer. Equal amount of protein samples (30 μg) were dissolved by 12% sodium dodecyl sulfate–polyacrylamide electrophoresis gels and transferred onto a polyvinylidene difluoride membrane. After being blocked with 5% nonfat milk in Tris buffered saline–Tween buffer, the membrane was probed with primary antibodies specific for phosphorylated and total ERK1/2 (CST), p38 (CST), JNK (CST), MMP-13 (Millipore), ADAMTS-5 (Abcam), and phosphorylated and total FGFR1 (Santa) followed by secondary antibodies. The signal was detected using chemiluminescent (Pierce) according to the manufacturer’s instruction. The antibody specific for β-actin (Sigma) was applied to normalize the protein expression levels.

Histological sections (3 μm thick) were deparaffinized and rehydrated. Afterwards, sections were heated in a citrate buffer solution for antigen retrieval and incubated with methanol and a 6% hydrogen peroxide solution to quench the endogenous peroxidase activity. The sections were incubated with 1% BSA (bovine serum albumin; Sigma-Aldrich) for 45 min to block nonspecific reactions. The sections were incubated with the following primary antibodies at the indicated dilutions overnight at 4°C: 1:50 SOX-5 (H-90 Santa Cruz Biotechnology), 1:200 MMP-13 (SAB4501900 Sigma-Aldrich), 1:200 MMP-8 (SAB4501895 Sigma-Aldrich), 1:100 IHH (AV45230 Sigma-Aldrich), and 1:250 Col2 (M2139 Santa Cruz Biotechnology). The Advanced HRP system (Dako, Carpinteria, Calif., USA) was used to detect antibodies and specimens were counterstained with Mayer’s hematoxylin, dehydrated and mounted for observation and quantification [18 (link)].
Quantification was performed using WCIF ImageJ software. Three sections from each sample were selected, and the surface, middle, and deep sections were obtained, calibrated and color deconvolution was performed. In Vectors, H DAB was chosen and the brown image was selected. After many tests, the Threshold interval was selected (0 to 180) and the colored Area Fraction was obtained and compared to the total area of each studied section.

The chondrocytes and knee articular cartilage tissue were flushed three times with PBS and lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. The samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim-milk powder for 30 min at room temperature and then incubated with the corresponding primary antibodies of NLRP3, ASC, caspase-1, IL-6, IL-18, TNF-α, MMP13, ADAMTS4/5, IGF-1, aggrecan and Col2a1 (1:1000, Sigma-Aldrich, St. Louis, MO) at 4 °C overnight. After washing with PBS, the membranes were incubated with horseradish peroxidase-labelled secondary antibody (1:2000, Cell Signaling Technology, Danvers, MA) for 1 h at room temperature. Ultimately, the intensity of protein expression was measured by an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Carlsbad, CA). GAPDH served as control.

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.