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Recombinant hmgb1

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Recombinant HMGB1 is a protein produced using recombinant DNA technology. HMGB1 is a nuclear protein that functions as a DNA-binding and architectural factor, involved in the regulation of gene expression.

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Lab products found in correlation

3 methyladenine 3 ma, by Merck Group (2 mentions) Phosphorylated p53, by Cell Signaling Technology (1 mentions) Mapk family antibody sampler kit, by Cell Signaling Technology (1 mentions) Bax antibody, by Cell Signaling Technology (1 mentions) P38 mapk inhibitor sb203580, by Selleck Chemicals (1 mentions) Tnf α, by Thermo Fisher Scientific (1 mentions) Phospho mapk family antibody sampler kit, by Cell Signaling Technology (1 mentions) Bcl 2, by Cell Signaling Technology (1 mentions) Picopump, by World Precision Instruments (1 mentions) Hydraulic micropositioner, by Kopf Instruments (1 mentions) Yohimbine, by Merck Group (1 mentions) Methyllycaconitine, by Merck Group (1 mentions) Imidazole, by Merck Group (1 mentions) Atipamezole, by Merck Group (1 mentions) Hmgb1, by Merck Group (1 mentions) Dexmedetomidine, by Merck Group (1 mentions) α actin, by Santa Cruz Biotechnology (1 mentions) Mouse monoclonal antibodies against cpla2, by Santa Cruz Biotechnology (1 mentions) Streptozotocin (stz), by Merck Group (1 mentions) Rabbit anti hmgb1 abs, by Abcam (1 mentions)

17 protocols using recombinant hmgb1

1

Intraspinal HMGB1 Injection in Mice

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Mice were anesthetized with a cocktail of ketamine/xylazine (80 and 10mg/kg, respectively). Using aseptic technique, a laminectomy was performed at the T12–13 vertebral level after which the spinal column was secured via the spinous processes adjacent to the laminectomy site using Adson forceps fixed in a spinal frame. Sterile glass micropipettes (pulled to an external diameter of ~25μm and pre-filled with sterile recombinant HMGB1 (R&D Systems; 500ng/mouse; n=6) or sterile PBS (n=6) were positioned at 0.4mm lateral from midline. From the meningeal surface, pipettes were lowered 0.8mm using a hydraulic micropositioner (David Kopf Instruments, Tujunga, CA). Using a PicoPump (World Precision Instruments, Sarasota, FL), 1μl of solution was injected over a period of 15min. To minimize fluid reflux, pipettes remained in place for 2 additional minutes to allow the injectate to dissipate into the parenchyma. To facilitate localization of the injection sites for anatomical analysis, a small amount of sterile charcoal was placed on the adjacent dura before closing the overlying tissues. Structural testing of recombinant HMGB1 by R&D Systems indicates that the recombinant protein does contain the disulfide bond at Cys23 and Cys45 as well as a free thiol at Cys106. This disulfide form of HMGB1 contains cytokine-stimulating activity (Yang et al., 2012 (link)).
Kigerl K.A., Lai W., Wallace L.M., Yang H, & Popovich P.G. (2017). High mobility group box-1 (HMGB1) is increased in injured mouse spinal cord and can elicit neurotoxic inflammation. Brain, behavior, and immunity, 72, 22-33.
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2

Apoptosis and Cytokine Signaling Assays

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Rat anti-mouse bcl-2 and Bim antibody were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Recombinant HMGB1 was purchased from R&D System, Minneapolis, MI. Rat anti-mouse p53, phosphorylated p53, bcl-2 and Bax antibody were purchased from Cell Signaling Technology, Beverly, MA. MAPK family antibody sampler kit and phospho-MAPK family antibody sampler kit were purchased from Cell Signaling Technology, Beverly, MA. Annexin V- fluorescein isothiocyante (FITC) was purchased from BD, San Diego, CA. The p38 MAPK inhibitor (SB203580) was purchased from Selleck Chemicals, Houston, TX. Enzyme-linked immunosorbent assay (ELISA) kits of IL-12, IL-2, IL-4, interferon (IFN)-γ, and TNF-α were purchased from Biosource, Worcester, MA. Nuclear extract and nuclear factor of activated T cell (NF-AT) assay kits were purchased from Active Motif, Carlsbad, CA.
Luan Y.Y., Jia M., Zhang H., Zhu F.J., Dong N., Feng Y.W., Wu M., Tong Y.L, & Yao Y.M. (2017). The potential mechanism of extracellular high mobility group box-1 protein mediated p53 expression in immune dysfunction of T lymphocytes. Oncotarget, 8(68), 112959-112971.
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3

Neutralizing Trauma-Released HMGB1 in Bone Fracture

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Fifty μg/kg (100 μl) of recombinant HMGB1 (R&D System, Minneapolis, MN) was administered intraperitoneally. To neutralize trauma-released HMGB1, 50 μg of anti-HMGB1 neutralizing monoclonal antibody (2G7, mouse IgG2b supplied by Dr. Tracey's Laboratory, Manhasset, NY) in 100 μl of saline was administered intraperitoneally, 60 min before bone fracture. Control animals received the same volume (100 μl) of the vehicle (saline).
Vacas S., Degos V., Tracey K.J, & Maze M. (2014). High-mobility Group Box 1 Protein Initiates Postoperative Cognitive Decline by Engaging Bone Marrow-derived Macrophages. Anesthesiology, 120(5), 1160-1167.
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4

Modulation of HMGB1-induced inflammation

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Recombinant HMGB1 (R&D System, Minneapolis, MN) was dissolved in phosphate-buffered saline and administered ip 50 μg/kg, a dose that we had earlier reported to produce a similar inflammatory and cognitive response as that seen after surgery.5 (link)Dexmedetomidine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% sterile saline and administered 50 μg/kg ip every 2 hours for three doses immediately following HMGB1 (Figure 1 A, B) or at 20 μg/kg ip for four doses in the surgical model (Figure 1 C, D). These doses were selected to simulate perioperative sedation either with (Figure 1 C, D) or without isoflurane anesthesia (Figure 1 A, B).
Yohimbine (Sigma-Aldrich) was dissolved in 0.9% sterile saline and 1.5 mg/kg was administered ip, a dose that effectively blocks α2 adrenoceptor-mediated responses.17 (link)Atipamezole (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 5% dimethyl sulfoxide in saline and 3 mg/kg was administered ip, a dose that effectively blocks both imidazole receptor and α2 adrenoceptor-mediated responses.
Methyllycaconitine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% sterile saline and 4mg/kg ip was administered, a dose that blocks α7 nicotinic acetylcholine receptor-mediated response.2 (link)
Hu J., Vacas S., Feng X., Lutrin D., Uchida Y., Lai I.K, & Maze M. (2018). Dexmedetomidine prevents cognitive decline by enhancing resolution of HMGB1-induced inflammation through a vagomimetic action in mice. Anesthesiology, 128(5), 921-931.
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5

Reagents and Antibodies for Cell Signaling

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All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), Life Technologies (Grand Island, NY), and Thermo Scientific (Rockford, IL), unless otherwise indicated. Recombinant HMGB-1 was purchased from R&D Systems (Minneapolis, MN) and IBL International Corp (Toronto, ON). Rabbit polyclonal antibody against von Willebrand factor, mouse monoclonal antibodies against cPLA2, α-actin and GAPDH were purchased from Santa Cruz (Santa Cruz, CA). Streptozotocin (STZ) was purchased from Sigma. All the other reagents were obtained from standard commercial suppliers unless otherwise noted.
Gong Y., Jin X., Wang Q.S., Wei S.H., Hou B.K., Li H.Y., Zhang M.N, & Li Z.H. (2014). The involvement of high mobility group 1 cytokine and phospholipases A2 in diabetic retinopathy. Lipids in Health and Disease, 13, 156.
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6

Investigating HIV-2 Replication in DCs

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Example 16

The question of the susceptibility of HIV-2-infected DCs on NK-dependent triggering of viral replication was addressed, similarly to HIV-1. The influence of HMGB1 in that process was evaluated.

1—Infection of DCs with HIV-2: iDCs were plated in 96-well culture plates at 500,000 cells/well and incubated for 3 hours at 37° C. in a 5% CO2 atmosphere with HIV-2 (20 ng p24/ml).

2—NK-DC cocultures: cells were harvested, washed three times with RPMI containing 10% FCS and, when indicated, aNK cells were added at a NK:DC ratio of 1:5. When indicated, recombinant HMGB1 was added at 10 μg/ml (R&D Systems), or rabbit anti-HMGB1 Abs (1 μg/ml) (Abcam, Cambridge, UK). NK-DC cocultures lasted 3 to 7 days before quantification of viral production in culture supernatants.

3—Quantification of HIV-2 viral production: the concentration of HIV-2 particles in the supernatants was determined with the p24 ELISA kit (Ingen, Belgium).

As shown on FIG. 6C, a very low level of HIV-2 production was detected after three days of infection, whether infected DCs were cultured alone or in the presence of aNK cells. rh-HMGB1 induced a slight increase in viral replication. As observed in our previous studies with HIV-1, day 3 of infection of DCS is too early to detect significant viral replication. These coculture supernatants will be tested again at day 7.

US10012655B2. Method for quantitating total HMGB1 protein in biological samples (2018-07-03). INSTITUT PASTEUR [FR]. Inventors: Marie-Lise Gougeon [FR], Hela Saidi [FR], Marie-Therese Melki [FR], Beatrice Poirier-Beaudoin [FR], Valerie Seffer [FR].
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7

Preparation and Characterization of ODSH

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ODSH (2-O, 3-O-desulfated heparin) was prepared in powder form by Scientific Protein Laboratories (Waunakee, WI) (Fryer et al. 1997 (link)). The stock solution was made in distilled water to obtain a concentration of 50 mg/ml for the in vitro experiments and 20 mg/kg for the in vivo experiments. The stock solution was further diluted to intended concentrations with either normal saline for in vivo studies or cell culture media for in vitro experiments. Recombinant HMGB1 (in the reduced form) was purchased from R&D Systems (Minneapolis, MN). Published studies suggest that rHMGB1 is in a fully reduced state and can bind to RAGE, TLR2 and TLR4, to induce inflammatory responses (Entezari et al. 2014 (link); Wang et al. 2019 (link)). Thus, the rHMGB1 used in this study was anticipated to be in the reduced form.
Wang M., Gauthier A.G., Kennedy T.P., Wang H., Velagapudi U.K., Talele T.T., Lin M., Wu J., Daley L., Yang X., Patel V., Mun S.S., Ashby CR J.r, & Mantell L.L. (2021). 2-O, 3-O desulfated heparin (ODSH) increases bacterial clearance and attenuates lung injury in cystic fibrosis by restoring HMGB1-compromised macrophage function. Molecular Medicine, 27, 79.
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8

Mouse Spleen Lymphocyte Separation and Analysis

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Mouse spleen lymphocyte separation medium and PBS were purchased from HaoYang Technology Co. Ltd., Tianjin, China. RPMI 1640 was purchased from Hong Wei Biotech Inc., Shijiazhuang, China. Fetal calf serum (FCS) was purchased from Gibco Co., CA, USA. The complete medium used throughout the experiments was RPMI 1640, supplemented with 10% FCS. Opti-MEM medium was purchased from Gibco Co. Recombinant HMGB1 was purchased from R&D Systems, Minneapolis, USA. CD11c+ (N418) MicroBeads were purchased from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. ConA was purchased from Sigma, St. Louis, MO. The antibodies used for flow cytometry analysis, including FITC anti-mouse MHC-II, PE anti-mouse CD86 antibody, and APC anti-mouse CD80 antibody were purchased from Miltenyi Biotec GmbH. ELISA kits for IL-12, TNF-α, IL-2, and IFN-γ were purchased from ExCell Biotech (Taicang) Co., Ltd., Shanghai, China. CCK-8 cell proliferation assay was purchased from Sigma, St. Louis, MO. The TRIzol reagent was purchased from Invitrogen, USA. The reverse transcription system were purchased from Promega, Madison, WI. miRNA first strand cDNA synthesis (Poly A Tailing) kit was purchased from Sangon Biotech, Shanghai, China. SYBR Green PCR Master MIX was purchased from KAPA Biosystems, Boston, MA. Lipofectamine 2000 was purchased from GenePharma Biotech Co., Ltd, China.
Zhu J., Wang F.L., Wang H.B., Dong N., Zhu X.M., Wu Y., Wang Y.T, & Yao Y.M. (2017). TNF-α mRNA is negatively regulated by microRNA-181a-5p in maturation of dendritic cells induced by high mobility group box-1 protein. Scientific Reports, 7, 12239.
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9

Recombinant HMGB1 Activation and Neutralization

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Recombinant HMGB1 was purchased from R&D Systems (Minneapolis, MN, USA). Stimulating activity of Recombinant HMGB1 was confirmed in mouse macrophages by assaying TNF release, with an ED50 of 3–12 μg/ml. Neutralizing anti-HMGB1 IgY was purchased from SHINO-TEST Corporation (Kanagawa, Japan) and control nonimmune IgY was purchased from Fitzgerald (North Acton, MA, USA). All other chemicals were obtained from Sigma-Aldrich, except where noted.
Yang J., Zhao Y., Zhang P., Li Y., Yang Y., Yang Y., Zhu J., Song X., Jiang G, & Fan J. (2016). Hemorrhagic shock primes for lung vascular endothelial cell pyroptosis: role in pulmonary inflammation following LPS. Cell Death & Disease, 7(9), e2363-.
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10

HMGB1 Induces ET-1 Release in HPAEC

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HPAEC (CC-2530; Lonza) were cultured in commercial HPAEC medium (EGM-2, Lonza) supplemented with penicillin (100 U/ml)–streptomycin (100 μg/ml), 10% fetal bovine serum (FBS) at 37oC in a humidified 5% CO2 incubator. All experiments were performed on endothelial cells within the fifth passage. HPAEC was seeded in 96-well plates (1×104 cells/well) for 24 hours, and recombinant HMGB1 (R&D systems) was treated to HPAEC in various concentrations (10, 20, 30, and 40 ng/ml) for 24 hours. To inhibit the ET-1 release induced by HMGB1, anti-HMGB1 antibody (ab18256; Abcam) (0.2 μg/ml, 1 μg/ml) and anti-RAGE antibody (ab3611; Abcam) (0.2 μg/ml, 1 μg/ml) were co-treated with recombinant HMGB1 of 40 ng/ml. The culture supernatant was harvested, and ET-1 release was measured using a commercial human ET-1 ELISA kit (#900-020A; Enzo Life Sciences Inc.) in accordance with the manufacturer’s instructions.
Yang P.S., Kim D.H., Lee Y.J., Lee S.E., Kang W.J., Chang H.J, & Shin J.S. (2014). Glycyrrhizin, inhibitor of high mobility group box-1, attenuates monocrotaline-induced pulmonary hypertension and vascular remodeling in rats. Respiratory Research, 15, 148.
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