A02802 1

Total protein was extracted from chondrocytes/cartilage tissue using radio-immunoprecipitation assay lysis buffer containing 1 mM phenylmethanesulfonyl fluoride. Protein concentrations were measured using a bicinchoninic acid assay. Equal amounts of protein (20 μg/lane) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk and incubated with primary antibodies against TLR4, Myd88, IKK-β, β-actin, aggrecan, MMP-3, MMP-9 (ab13556, ab2064, ab124957, ab8227, ab36861, ab52915, ab76003, AbCam, USA, 1:1000, respectively), MMP-13 (ab39012, AbCam, USA, 1:5000), ADAMTS-4 (ab150370, AbCam, USA, 1:1000), ADAMTS-5 (A02802-1, Boster, China, 1:500), CGRP (14959 S, Cell Signaling Technology, USA, 1:1000), SP (S8305, Sigma, USA, 1:2000), NF-κB p65 (ab16502, AbCam, USA, 1:5000), and GAPDH (ab181602, AbCam, USA, 1:10,000) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody IgG (ab6721, AbCam, USA) at room temperature. Immune reactivity was visualized using an enhanced chemiluminescent reagent (Pierce; Thermo Fisher Scientific, Inc.). Band intensity was quantitatively analyzed by densitometric analysis on Image J software version 1.37 (National Institutes of Health, Bethesda, MD, USA).