Anti adamts5

Control and treated chondrocytes were lysed, followed by SDS-PAGE electrophoresis as previously described [13 ]. The primary antibodies used were listed: anti-iNOS, anti-COX-2, anti-β-actin, and HRP-conjugated secondary antibody from Santa Cruz Biotechnology, Santa Cruz, CA; and anti-ADAMTS-4, anti-ADAMTS-5, anti-aggrecan, anti-collagen II, anti-p65, anti-p-p65, anti-p-IκBα, and anti-IκBα from Abcam. Finally, the bands were visualized with the ECL reagent.

For immunohistochemistry, the sections were blocked with 10% goat serum and incubated with the antibodies for anti-ADAMTS-5 (ab41037, Abcam), overnight at 4°C. Then, the sections were incubated with the appropriate secondary antibodies, and the nuclei were stained with DAPI solution at room temperature. Finally, the sections were visualized with diaminobenzidine (DAB) (Gene Tech, Shanghai, China) and viewed under a light microscope (Nikon H550L, Tokyo, Japan).
For immunofluorescence analysis, the sections were blocked with 10% goat serum and incubated with anti-ADAMTS-5 (Abcam), anti-CD31 (R&D Systems), anti-CD68 (R&D Systems), and anti-α-smooth muscle cell (α-SMA, GeneTex) antibodies overnight at 4°C. Then, the sections were incubated with two different IRDye® 800CW-conjugated 169 secondary antibodies. Finally, fluorescence images were captured with an OLYMPUS DX51 fluorescence microscope.

Stimulated NP cells were collected and then lysed with RIPA lysis buffer (CWBIO, Beijing, China) for total protein, or with an extraction kit for cytoplasmic and nuclear protein (CWBIO). After being centrifuged and collected, the supernatant containing proteins was then quantified using a BCA assay kit (CWBIO). Afterwards, the proteins were separated by SDS-PAGE and electro-transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). After being blocked with 5% bovine serum albumin in TBST for 1 h, the membranes were then incubated at 4°C overnight with anti-CBX4 (1:1000; Abcam), anti-MMP3 (1:1000; Abcam), anti-MMP13 (1:1000; Abcam), anti-ADAMTS5 (1:1000; Abcam), anti-COX2 (1:1000; Abcam), anti-COL2A1 (1:1000; Abcam), anti-P53 (1:1000; Abcam), anti-P21 (1:1000; Abcam), anti-β-actin (1:1000; Abclonal, Wuhan, China), anti-p-p65 (1:1000; Abclonal), anti-p65 (1:1000; Abclonal), or anti-Histone H3 (1:1000; CST, Danvers, USA) antibodies. Afterwards, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:8000; Abclonal). The protein bands were visualized using an Enhanced Chemiluminescence kit (Vazyme, Nanjing, China) and then quantified by Image J (National Institutes of Health, Bethesda, USA).

The total protein of CHON-001 cells was isolated by RIPA buffer (Beyotime). Then, the crude extracted proteins were further subdivided by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE, Beyotime) and polyvinylidene difluoride membranes (PVDF, Millipore, Danvers, MA, USA). Following, the membranes were blocked by non-fat milk and washed by 1 × Tris-buffered saline tween-20 (TBST, Sigma-Aldrich). Afterwards, the membranes containing the proteins were incubated with the primary antibodies, including anti-MMP13 (1: 3,000, Abcam, Cambridge, UK), anti-ADAMTS5 (1: 250, Abcam), anti-GAPDH (1: 2,500, Abcam), and anti-Aggrecan (1: 100, Abcam), and then incubated with the second antibodies anti-rabbit IgG (1: 20,000, Abcam) or anti-mouse IgG (1: 2,000, Abcam). The result was detected by Electrochemiluminescence (ECL) detection kit (Roche Diagnostics GmbH, Mannheim, Germany).

Primary antibodies were rat monoclonal antibody directed against GAL3 kindly provided by Dr. HakonLeffler (Lund University, Sweden), anti-VDIPEN IgG by Irwin Singer and Ellen Bayne (Merck Research Laboratories, Rahway, NJ, USA), mouse monoclonal anti-acetylated α-tubulin and rabbit polyclonal anti-γ-tubulin from Sigma-Aldrich (Saint-Louis, MO, USA), mouse monoclonal anti-cytochrome C (Bd Pharmingen, Le pont de Claix, France), mouse monoclonal anti-type X collagen (Diagomics, Blagnac, France), rabbit polyclonal anti-ADAMTS-5 (Abcam, Cambridge, UK), mouse monoclonal anti-type 2 collagen (Abcam), and rabbit polyclonal anti-aggrecan (Millipore, Guyancourt, France). Secondary antibodies were goat anti-mouse Alexa-488 and goat anti-rabbit Alexa-568 (Life Technologies, Paisley, UK).