Human or mouse NP cells were lysed with a radioimmunoprecipitation assay buffer (Beyotime, China) to extract cellular proteins. The proteins were separated using 10% SDS-PAGE. The separated proteins were transferred from the gel to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) and blocked with skimmed milk powder for 1 h. Subsequently, the membrane was immersed in the relevant protein antibody solution (collagen II, aggrecan, SOX9, MMP3, MMP13, ADAMTS4, or ADAMTS-5; 1:1000; Abcam) at 4 °C for 8 h. After washing away excess antibody, the membrane was incubated with a secondary antibody at room temperature (25 °C) for 2 h. The protein bands were observed with an Amersham lmager 600 (General Electric Company, USA) and analyzed with the ImageJ software (National Institute of Health, Bethesda, MD, USA).
Adamts4
Manufactured by Abcam
Sourced in United States, United Kingdom
ADAMTS4 is a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of enzymes. It is a zinc-dependent metalloproteinase that can cleave and degrade the extracellular matrix component aggrecan.
Automatically generated - may contain errors
Lab products found in correlation
Mmp13, by Abcam (16 mentions)
Aggrecan, by Abcam (10 mentions)
Adamts5, by Abcam (10 mentions)
Collagen 2, by Abcam (6 mentions)
β actin, by Abcam (5 mentions)
Mmp 9, by Abcam (4 mentions)
Nf κb p65, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Mmp13, by GeneTex (3 mentions)
Bcl 2, by Abcam (3 mentions)
Gapdh, by Abcam (3 mentions)
Tnf α, by Abcam (3 mentions)
Bcl2 associated x (bax), by Abcam (3 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Mmp 1, by Abcam (2 mentions)
Bx53 microscope, by Olympus (2 mentions)
Image pro plus version 6, by Media Cybernetics (2 mentions)
Phospho nf κb p65 ser536, by Cell Signaling Technology (2 mentions)
Sirt6, by Abcam (2 mentions)
Col2a1, by Abcam (2 mentions)
26 protocols using adamts4
1
Western Blot Analysis of Chondrocyte Markers
2
Protein Extraction and Western Blotting
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Total protein was extracted using RIPA Lysis Buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor and phosphatase inhibitor (Bimake, TX, USA). Equal amounts of protein samples were subjected to 10% or 15% SDS-PAGE (Solarbio, Beijing, China), separated by electrophoresis, and transferred to nitrocellulose filter membranes. The membranes were blocked with 5% skim milk powder for 1 h at room temperature, and then, the target membranes were incubated at 4°C overnight with primary antibodies against COL2A1, ACAN, ADAMTS4, ADAMTS5, MMP3, MMP13, p16 or p21 (1 : 1000, Abcam), ADAMTS5 (1: 250, Abcam), MMP9, iNOS, COX2, NF-κB p65, p-NF-κB p65, AKT, p-AKT, p-PI3K, PI3K, p-IKBα, IKBα, p-IKK or IKK (1 : 1000, Cell Signaling Technology), or GAPDH (1 : 5000, Proteintech). After incubated with HRP-conjugated secondary antibody for 1 h at room temperature, the signal was developed using an ECL chemiluminescence detection kit (Beyotime, Shanghai, China), and images were captured on ImageQuant Las4000mini (GE Healthcare, Tokyo, Japan).
3
Chondroprotective Effects of Silibinin
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Silibinin (purity >98 %), recombinant human IL-1β, collagenase type II, Safranin-O/Fast Green and dimethylsulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cell-Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Primary antibodies against COX-2, iNOS, MMP-1, MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 were purchased from Abcam (Cambridge, MA, USA). Primary antibodies against p65, p-p65, IkBα, p-IkBα, PI3K, p-PI3K, Akt and p-Akt were purchased from Cell Signaling Techonology (Beverly, MA, USA). Goat anti-rabbit and goat anti-mouse horseradish peroxidase conjugates were purchased from Bio-Rad Laboratories (Calif., USA). Fetal bovine serum (FBS), bovine serum albumin (BSA), Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium and 0.25% trypsin-ethylendiaminetetraacetic acid (trypsin–EDTA) were purchased from Gibco (Life Technologies Corp. Carlsbad, Calif., USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, Calif., USA). QuantiTect Reverse Transcription kit was purchased from Qiagen (Valencia, CA). SYBR Green Master Mix was purchased from Bio-Rad Laboratories (Calif., USA). ELISA kits of PGE2, TNF-α and IL-6 were purchased from R&D systems (Minneapolis, MN, USA). Griess reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals were of reagent grade.
4
Immunohistochemical Analysis of Cartilage Proteins
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Immunohistochemical analyses were performed to detect the expression levels of aggrecan (1: 500; GeneTex, Inc., USA), collagen II (Col-II) (1: 100; II-II6B3 deposited in DSHB by T.F. Linsenmayer), caspase-3 (1: 200; Boster Co., China), matrix metalloproteinase (MMP)-13 (1: 200; GeneTex, Inc., USA), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 (1: 200; Abcam, Inc., USA) in cartilage. Paraffin sections were deparaffinized with xylene and rehydrated with ethanol. After antigen retrieval with 0.05% trypsin and inactivation of endogenous peroxidases with 0.3% H2O2, the sections were incubated at 4°C overnight with the target protein antibody. The remaining experimental procedures were conducted in accordance with the protocols provided with the PV-6000 DAB detection kit and ZLI-9018 DAB kit (both from ZSGBBIO Corp., China), before being counterstained with hematoxylin. Images were captured at 100× magnification using a BX53 microscope (Olympus, Japan) and semiquantitatively analyzed with Image-Pro Plus version 6.0 software (Media Cybernetics, USA). The average optical density intensity of each protein, expressed as the integrated optical density/mm2, was defined as the value of the integrated optical density divided by the cartilage area.
5
Protein Expression Analysis in NP Cells
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NP cells were harvested and lysed in radioimmunoprecipitation assay buffer. Protein concentrations were determined with the bicinchoninic acid assay (Beyotime, Shanghai, China). Proteins were separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, which was blocked with 5% non-fat milk and then incubated overnight at 4°C with antibodies against the following proteins: SOX9 (1:1000), type II collagen (1:1000), aggrecan (1:1000), MMP3 (1:1000), MMP13 (1:4000), ADAMTS4 (1:1000), ADAMTS5 (1:500), Bcl-2 (1:1000), and β-actin (1:2000) (all from Abcam, Cambridge, MA, USA); and phospho-NF-κB p65 (Ser536) (1:2000) and NF-κB p65 (1:1000) (Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated for 2 h at 37°C with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Abcam). Protein bands were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). β-actin was used as a loading control. The experiment was repeated three times.
6
Protein Expression Analysis in Cells
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Total proteins of the cells were extracted with RIPA (Biocolors Biotechnology, China) lysis buffer, and protein concentrations were determined using a Pierce BCA Protein assay kit (Pierce Biotechnology, USA). Protein samples (30 µg/lane) were separated on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). After transfer, the membrane was blocked with 5% non-fat milk for one hour at room temperature, and incubated with the following primary antibodies (BioTeke, China) at 4°C overnight: SIRT6 (1:500), aggrecan (1:500), MMP-13 (1:500), ADAMTS4 (1:500), COL2A1 (1:1000), and GAPDH (1:1,000; all Abcam, UK). Then, the membranes were incubated with goat anti-rabbit IgG secondary antibody, and signals were detected by chemiluminescence.37 GAPDH was used as a reference. All bands were detected using ECL Western Blot Kit (Amersham Biosciences, UK) following the manufacturer’s protocol.
7
Antibody Detection Protocol for Osteoarthritis
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Antibodies against GAPDH, COL2A1, ACAN, ADAMTS4, ADAMTS5, MMP9, MMP13, TNF-α, NOX1, NOX2, iNOS, COX2, and NLRP3 were purchased from Abcam Inc. Catalase, SOD1, BAX, BCL2, Caspase3, Cleaved-Caspase3, TAK1/NF-κB signaling proteins (TAK1, phosphor-TAK1, P65, phosphor-P65, IκBα, and phosphor-IκBα), and IgG secondary antibodies were purchased from Cell Signaling Technology Inc. Antibody against GAPDH was from Proteintech Group Inc. Aloin reagent was from MedChemExpress, and human TNF-α was from R&D Systems. Abbreviations and descriptions were shown in Supplement Table 1 .
8
Investigating Inflammatory Mediator Modulation
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TNF-α (Catalog #: ab6671), Collagen 10 (Catalog #: ab49945), MMP3 (Catalog #: ab52915), MMP13 (Catalog #: ab39012), iNOS (Catalog #: ab15323), UBA1 (Catalog #: ab34711) Adamts4 (Catalog #: ab1855722) and Adamts5 (Catalog #: ab41037) were purchased from Abcam (MA, USA), Adalimumab (Catalog #: A2010) and Glucosamine (Catalog #: S6400) were purchased from Selleckchem (TX, USA), UBE2N (Catalog #: 6999), phospho-RIP1 (Catalog #: 65746), NF-κB phospho-p65 (Catalog #: 3033), NF-κB p65 (Catalog #: 8242), RIP1 (Catalog #: 3943), Anti-rabbit IgG (H + L) Alexa Fluor 555 (Catalog #: 4413) and Anti-mouse IgG (H + L) Alexa Fluor 488 (Catalog #: 4408) were purchased from Cell Signaling Technology (MA, USA), Vimentin (Catalog #: SC6260), HA-Tag (Catalog #: SC7392), IκB (Catalog #: SC1643) and phospho-IκB (Catalog #: SC8404) were purchased from Santa Cruz Biotechnology (CA, USA), Spermidine (Catalog #: 05292) and Aggrecan (Catalog #: AB1031) were purchased from Sigma (MO, USA), CLYD (Catalog #: 1110-1-AP) and TRIM21 (Catalog #: 12108-1-AP) were purchased from PeproTech (NJ, USA).
9
Protein Expression Analysis of Cellular Markers
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We followed the methods of Sun et al. Briefly, total protein of cells was harvested using RIPA lysis buffer (Beyotime). Protein extracts were separated by using SDS-PAGE gels, followed by the transfer to a PVDF membrane. After blocking with 5% nonfat milk, the bands were incubated with indicated primary antibodies (Tsg101, Cell Signaling Technology; CD63, Abcam; CD9, Abcam; GM130, Abcam; NAMPT, Adipogen; P16, Abcam; MMP-3, Abcam; ADAMTS-4, Abcam; Aggrecan, Abcam; Collagen II Abcam; Sirt1, Abcam; Sirt3, Abcam; Sirt5, Abcam; β-actin, Abcam) overnight under 4°C. Then, the bands were incubated with horseradish peroxidase-linked anti-mouse IgG (Cell Signaling Technology) or anti-rabbit IgG (Abcam) secondary antibody for 1 h. The bands were exposed with enhanced chemiluminescence (ECL, Thermo Fisher Scientific).
10
Protein Extraction and Western Blot Analysis
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The transfected SW1353 cells were centrifuged for 5 min after adding cold PBS and washed for three times with PBS. Then they were cleaved at 4°C for 30 min with Nuclear and Cytoplasmic Protein Extraction Kit (China Haimen Beishi Biotechnology Research Institute), and placed at 4°C for 10 min. The protein was quantified with BCA protein detection kit (Beyotime Institute of Biotechnology). A total of 20 μG protein/lane was separated by 10% SDS/PAGE and transferred to PVDF membrane. The membrane was sealed overnight at 4°C with 5% skimmed milk powder, and then it was incubated with anti-SIRT1 (1:1000), collagen II (1:1000), aggrecan (1:1000), MMP13 (1:1000), ADAMTS4 (1:1000) and GAPDH (1:1000) (Abcam, Cambridge, MA, U.S.A.) were added. After incubating overnight, it was incubated with 1:5000 labeled anti-rabbit secondary antibody for 1 h. After that, the gray values of the target bands and the internal reference bands were recorded by ECL chemiluminescence. The experiment was conducted according to the literature method [25 (link)].
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