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4 protocols using antibodies against hmgb1

1

Fenofibrate and LPS Regulation of HMGB1 and PPAR-alpha

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Fenofibrate (catalog: F6020) and LPS (catalog: L3755) were purchased from Sigma-Aldrich (St. Louis, MO). All of the cell culture media and supplements were from Sigma. Antibodies against HMGB1 (catalog: 3935) were from Cell Signaling Technology (Danvers, MA). The anti-PPARα antibody (catalog: ab8934) and anti-histone H3 antibody (catalog: ab1791) were obtained from Abcam (Hong Kong). The anti-actin antibody (catalog: A2668) was obtained from Sigma. Primers for quantitative real-time reverse transcriptase-PCR were designed using GenBank sequences.
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2

Mitochondrial Function and Oxidative Stress Assays

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MitoTracker Green, MitoTracker DeepRed, MitoSox, and CellRox were purchased from Molecular Probes. Proteinase K was purchased from Roche. Recombinant human GM-CSF was purchased from BD. Recombinant human LL-37, R837, ODN-TTAGGG, and ODN-2216 were purchased from InvivoGen. FcR Blocking Reagent was purchased from Miltenyi Biotec. Antibody against 8OHdG (J-1; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology, Inc. Antibodies against LL-37, TFAM (Clone 18G102B2E11), Mitofilin (Clone 2E4AD5), γH2A.X, TOMM20 (Clone 4F3), Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3-phosphate dehydrogenase (GADPH), dsDNA, LAMP1 (Clone H4A3), and Rab7 were purchased from Abcam. Antibodies against MnSOD were purchased from Millipore. Antibodies against HMGB1, PGC1α, and Phospho-(Ser/Thr) PKA Substrate were purchased from Cell Signaling Technologies. Genomic DNA was obtained from BioChain. IRS661 and DVX41 were a gift from Dynavax Technologies Corporation. Specified neutrophils were cultured in the presence of αRNP-IgG (50 µg/ml), Recombinant human GM-CSF (50 ng/ml), diphenylene iodonium (DPI; 10 µM), MT (10 µM), CCCP (25 µM), Rotenone (5 µM), Oligomycin (10 µM) plus Antimycin (1 µM; O+A), IRS661 (TLR7 antagonist; 1 µM), DVX42 (TLR8 antagonist; 1 µM), Bafilomycin A1 (BafA1; 1 µM), 8Br-cAMP (100 µM), IBMX (50 µM), or H89 (1 µM).
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3

Investigating Inflammation Pathways with Reagents

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EP (Ethyl pyruvate) and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, United States). TAK-242 and PPARγ agonist Rosiglitazone (RSG) were provided by MedChemExpress Company (Princeton, NJ, United States). Antibodies against HMGB1, TLR4, and IL-1β, were from Cell Signaling Biotechnology (Danvers, MA, United States). Antibody against CD68 were from Novus Biologicals (United States). Antibodies against PPARγ, LXRα, and ABCA1 were obtained from Abcam Company (Cambridge, United Kingdom). Rabbit Antibody against actin was provided by Proteintech Group, Inc. (Chicago, IL, United States). Corticosterone, IL-1β, and TNF-α ELISA kits were obtained from Westang Company (Shanghai, China). HMGB1 parameter assay kits were purchased from Cusabio Biotech, Co., Ltd. (Wuhan, China).
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4

Protein and Stem Cell Marker Analysis

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Protein concentrations were determined by bicinchoninic acid (BCA) method. Western blotting assay was performed as described previously [43 ]. Antibody against TLR4 was purchased from Abcam Company (Cambridge, USA). Antibodies against TLR2, GAPDH, Oct4, Sox2 and Nanog were purchased from Santa Cruz Biotechnology (USA). Antibodies against HMGB1, CD133, Sox2, CD44, c-myc, GSK3β, p-GSK3β, β-catenin, Myc, and Flag were obtained from Cell Signaling Technology, Inc. (USA). Secondary antibody (either anti-rabbit or anti-mouse) were purchased from Boster Biotechnology Company (China).
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