HMGB1 Pf4, HMGB1 Flox, and C57BL/6J male mice, littermates, age 8–12 weeks, were utilized in all experiments. DVT was induced using a validated model of venous stasis, inferior vena cava (IVC) ligation18 (link). Briefly, mice were anesthetized, underwent a midline laparotomy, the IVC is ligated completely with 6-0 nonabsorbable suture at the level of the renal veins, all visible side branches are ligated with 6-0 nonabsorbable sutures as well. Mice were then allowed to recover and later were sacrificed at 24 h (acute model) or 7 days (subacute/chronic) following IVC ligation. Thrombi, which included the thrombus and vessel wall, were excised and weighed immediately. Clots were processed for protein harvest or immunofluorescent (IF) staining. Where noted mice received recombinant HMGB1 (1 μg/g body weight, R&D Systems), the PAD4 inhibitor GSK199 (30 mg/kg, Cayman Chemical), or DNase I (100 units, ThermoFischer) via tail vein injection 30 min prior to IVC ligation.
Gsk199
Manufactured by Cayman Chemical
GSK199 is a laboratory reagent produced by Cayman Chemical. It functions as a potent and selective inhibitor of the RIPK1 kinase. GSK199 demonstrates nanomolar affinity for RIPK1 and displays high selectivity over related kinases.
Automatically generated - may contain errors
Lab products found in correlation
Afm30a, by Cayman Chemical (2 mentions)
Cay10723, by Cayman Chemical (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Hmgb1, by R&D Systems (1 mentions)
Cl amidine, by Cayman Chemical (1 mentions)
Gsk484, by Cayman Chemical (1 mentions)
Pyrocatechol, by Merck Group (1 mentions)
Foscarnet, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Hemavet hematology analyzer, by Drew Scientific (1 mentions)
5 protocols using gsk199
1
Murine Model of Deep Vein Thrombosis
2
Inhibition of Neutrophil NETosis
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Neutrophils were pre-treated with 10 μM diphenyleneiodonium chloride (DPI, Sigma Aldrich), pyrocatechol (100 μM, Sigma Aldrich), PAD inhibitors Cl-amidine (200 μM, Cayman Chemicals), GSK199 (10 μM, Cayman Chemicals), and GSK484 (10 μM, Cayman Chemicals) or DMSO control (0.01%) for 30 min at 37°C prior to treatment with PMA (100 nM) or different P. aeruginosa strains (MOI 10). Extracellular DNA was measured using Sytox Green as described above.
3
Inhibition of PAD Enzymes Impacts Viral Infection
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The PAD inhibitors Cl-amidine (Cl-A), BB-Cl-amidine (BB-Cl), GSK199, CAY10727, and AFM30a—also known as CAY10723—were obtained from Cayman Chemical (Ann Arbor). CHX and foscarnet (phosphonoformic acid, PFA) were from Sigma-Aldrich. All these compounds were reconstituted in dimethyl sulfoxide (DMSO) or ethanol accordingly to their solubility. HF4 and Thapsigargin were kindly provided by Dr. P. Thompson and Dr. M. Corazzari, respectively. Immediately before use, the inhibitors were diluted in the cell medium to their final concentrations. For the PADs inhibitors experiments, cells were pre-treated with the inhibitors for 1 h and then infected (MOI 1) by adding the virus without changing the medium. Following virus adsorption (2 h), the viral inoculum was removed, and a new medium with fresh inhibitor was added. After that, no more medium or inhibitor was added until the samples were collected.
4
Modulating Immune Responses in Stroke
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Platelets were depleted by i.v. injection of 2 mg/kg platelet-depleting antibodies (R300, Emfret) immediately after stroke induction. Platelet counts were measured using a Hemavet hematology analyzer (Drew Scientific). Neutrophils were depleted by i.p. injection of 5 mg/kg anti–mouse Ly6G (1A8, BE0074-1, Bio X Cell) and 5 mg/kg anti–rat Kappa IgG (MAR 18.5, BE0122, Bio X Cell) (22 (link)), 24 hours before stroke induction. Neutrophil depletion was confirmed via flow cytometry using granularity and Ly6C staining (Supplemental Figure 10 and ref. 22 (link)). Recombinant disulfide HMGB1 (rHMGB1; HM-120, HMGBiotech) was administered at 0.5 mg/kg i.v. 1 hour after stroke onset. HMGB1 was blocked by injection of 15 mg/kg of BoxA (HM-014, HMGBiotech) immediately before stroke induction. Neonatal NET-inhibitory factor (nNIF) and its inactive, scrambled peptide control (SCR) were synthesized as previously described by the University of Utah DNA/Peptide Synthesis Core Facility and injected i.v. at a concentration of 10 mg/kg at the indicated time points. PAD4 was inhibited by i.v. injection of GSK-199 (17489, Cayman Chemical) at 30 mg/kg, and NETs were degraded by i.v. injection of DNase I (dornase alfa, University of Utah Pharmacy) at 2.5 mg/kg, immediately before stroke induction.
5
Preparation and Dilution of PAD Inhibitors
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The PAD inhibitors Cl-A, BB-Cl, GSK199, and AFM30a—also known as CAY10723—were purchased from Cayman Chemical (Ann Arbor). All the compounds were solubilized in DMSO according to the manufacturer's instructions. Immediately before use, the inhibitors were diluted in the culture medium to the desired concentrations (Table S1 ).
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