The repertoire of MMP gene expression by chondrocytes was previously reported.29 (link),30 (link) In that regard, IL-1β a known activator of MMP gene expression for cultured human chondrocytes9 (link) regulated, in part, via activation of SAPK/MAPK signaling was employed to determine the extent to which the MMP1, 2, 3, 9 and 13 were expressed by C28/I2 chondrocytes in response to the addition of IL-1β (Cell Signaling Technologies).
Il 1β
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany
IL-1β is a cytokine that plays a crucial role in the inflammatory response. It functions as a mediator of the innate immune system.
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Lab products found in correlation
Tnf α, by Cell Signaling Technology (54 mentions)
Nlrp3, by Cell Signaling Technology (48 mentions)
Gapdh, by Cell Signaling Technology (36 mentions)
Caspase 1, by Cell Signaling Technology (35 mentions)
β actin, by Cell Signaling Technology (35 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (31 mentions)
Nf κb, by Cell Signaling Technology (16 mentions)
Bcl 2, by Cell Signaling Technology (16 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (14 mentions)
Lipopolysaccharides (lps), by Merck Group (13 mentions)
β actin, by Merck Group (12 mentions)
Caspase 1, by Abcam (12 mentions)
P nf κb, by Cell Signaling Technology (11 mentions)
Cleaved caspase 1, by Cell Signaling Technology (11 mentions)
Nf κb p65, by Cell Signaling Technology (11 mentions)
Caspase 1, by Santa Cruz Biotechnology (10 mentions)
Cleaved caspase 3, by Cell Signaling Technology (10 mentions)
Caspase 1, by Novus Biologicals (10 mentions)
Il 18, by Cell Signaling Technology (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
253 protocols using il 1β
1
Chondrocyte MMP Expression Regulation
2
Western Blot Analysis of Inflammasome Activation
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Western blots were performed as previously described59 (link). Briefly, lung tissues or cells were solubilized in lysis buffer containing protease inhibitor. Twenty μg of total protein was loaded on 12% SDS-PAGE, transferred to PVDF membrane, and probed with primary antibodies against Cav1 (Cell Signaling Technology, Danvers, MA), IL-1β (Cell Signaling Technology), caspase-1 (Santa Cruz Biotechnology, Dallas, TX) or β-actin (Sigma-Aldrich, St. Louis, MO). To detect inflammasome activation in cells, supernatants were collected and precipitated as described previously60 (link). Supernatants were precipitated with 1 volume methanol, ¼ volume chloroform, and the precipitate was washed in 1 volume methanol and resuspended in 50 μl SDS loading buffer followed electrophoresis and transferring as above. Proteins were probed with primary antibodies against IL-1β and caspase-1. Data were analyzed using NIH Image J software.
3
Analyzing Protein Expression and Processing
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To analyze protein expression and processing, cells were lysed directly with 1x SDS/PAGE sample buffer. Secreted proteins were isolated from cell supernatants by centrifugation at 2000 rpm for 10 minutes to remove cellular debris, followed by precipitation using trichloroacetic acid (TCA) overnight. Precipitated protein was pelleted by spinning at 13,000 rpm for 15 minutes at 4°C, then washed with ice-cold acetone, centrifuged at 13,000 rpm again for 10 minutes, before finally being suspended in 1x SDS/PAGE sample buffer. Samples were heated at 100°C for 5 minutes and then separated by SDS/PAGE and transferred to PVDF membranes (Millipore). Membranes were then probed with primary antibodies specific for murine caspase-11 (#C1354; Sigma-Aldrich), caspase-3 (#9662; Cell Signaling), caspase-8 (#4798; Cell Signaling), gasdermin D (#G7422; Sigma-Aldrich), IL-1β (12242S; Cell Signaling), and β-actin (#4967; Cell Signaling). Membranes were then probed with secondary antibodies anti-rat IgG (7077S; Cell Signaling), anti-mouse IgG (7076S; Cell Signaling), or anti-rabbit IgG (7074S; Cell Signaling). ECL Western Blotting Substrate and SuperSignal West Femto Substrate (Thermo Scientific) were used.
4
Investigating Inflammatory Signaling Pathways
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Recombinant IL-1β, forskolin and the inhibitors LY294002, U0126, SB203580 and SP600125 were obtained from Sigma-Aldrich Corp. Antibodies specific for β-actin, AKT, p-AKT (Ser 473), ERK1/2, p-ERK1/2 (Thr 202/Tyr 204), p38, p-p38 (Thr 180/Tyr 182), c-Jun, p-c-Jun (Ser 63), NF-κB, p-NF-κB (Ser 536), p-NF-κB (Ser 276), IL-1β and siRNAs targeting AKT, ERK1/2, p38 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Quinazoline (QNZ) was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The IL-1β and cAMP enzyme immunoassay kits were from Cayman Chemical (Ann Arbor, MI). Recombinant human MMP-7 was obtained from R&D systems (Minneapolis, MN, USA). MMP-7 cDNA plasmids were purchased from Origene Technologies (Rockville, MD, USA). All reagents for qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories. All other reagents were from Invitrogen (Carlsbad, CA) unless otherwise specified.
5
Quantitative Analysis of Skin Inflammasome
6
Xanthohumol Protects Against Oxidative Stress
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Xanthohumol (Xn), purity >98%, was provided by the Chengdu Herbpurify CO., LTD (Chengdu, China). LPS (Escherichia coli 055:B5) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C and Brusatol (specific inhibitors of the AMPK, and Nrf2, respectively), and tert-butyl hydroperoxide (t-BHP) were offered by Sigma-Aldrich (St. Louis, MO). Penicillin and streptomycin, Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were acquired from Invitrogen-Gibco (Grand Island, NY). Antibodies against Nrf2, Keap1, GCLC, HO-1, NQO1, GCLM, Trx-1, Txnip, NLRP3, casapase-1, ASC, IL-1β, Lamin B and β-actin were supplied by Cell Signaling (Boston, MA, USA) or Abcam (Cambridge, MA, USA). The Annexin V fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) apoptosis kit were offered from Beyotime Institute of Biotechnology (Jinlin, China). In addition, GSH, MDA, SOD and MPO test kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were offered by Sigma-Aldrich (St. Louis, MO, USA), if not otherwise indicated.
7
NLRP3 Inflammasome Activation Pathway
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Total protein from cell monolayers was harvested into M-PER lysis buffer (Thermo Fisher Scientific) and western blotting was performed to determine the expression of NLRP3 (Enzo life sciences), caspase-1, ASC, IL-1β (Cell Signaling Technologies) and IL-18 (MBL). Co-immunoprecipitation (Co-IP) was performed overnight at 4°C using NLRP3 antibody. Immunocomplexes were collected with Protein G Sepharose beads (GE Healthcare) for 1 h at 4°C prior to western blotting.
8
Quantification of IL-1β and IL-1RA in Cortical Tissue
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10 % cortical tissues (w/v) were homogenized on ice in lysis buffer with protease inhibitors (Roche), followed by centrifugation at 500 rpm for 1 min. The supernatant was collected and protein concentrations were adjusted by the bicinchoninic acid assay to ensure the same amount of protein being loaded for each sample (80 µg). Samples were mixed with SDS-containing sample buffers and incubated at 100 °C for 20 min. After electrophoresed on 12 % sample gel, proteins were transferred into polyvinylidene difluoride membrane (Millipore). The primary antibodies against IL-1β (Cell signaling), IL-1 RA (Thermo Scientific) and tubulin (Santa Cruz) were used at 1: 1000 concentrations for immunoblotting. Protein bands were quantified by ImageJ.
9
Murine Models for Metabolic Disorders
10
Western Blot Analysis of Inflammatory Markers
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In brief, total protein was isolated with RIPA lysis buffer at 14 000 g for 15 min at 4°C. The proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% dry nonfat milk in PBST for 1 h and incubated with primary antibodies against TNF-α (cat no. 3707; Cell Signaling Technology, Inc.; dilution, 1: 1000), IL-1β (cat no. 12703; Cell Signaling Technology, Inc.; dilution, 1: 1,000), IL-6 (ab6672; Abcam, USA; dilution, 1: 1000), netrin-1 (ab126729; Abcam, USA; dilution, 1: 1000), UNC5B (ab104871; Abcam, USA; dilution, 1: 500), and GAPDH (cat no. 5174; Cell Signaling Technology, Inc.; dilution, 1: 1000) overnight at 4°C and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 37°C. Finally, membranes were washed with PBST and the protein bands were detected using an ECL Western blot kit (Amersham Biosciences, UK).
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