LCA (cat # L6250; Sigma-Aldrich, St. Louis, MI, USA) was dissolved in DMSO at a stock concentration of 100 mM. LCA was used at a concentration of 0.03 µM, corresponding to normal human serum concentration [7 (link), 47 , 48 (link)]. Non-treated cells received 0.001% DMSO in the medium as a vehicle. Glutathione (GSH; cat # G4251; Sigma-Aldrich) was used at a final concentration of 5 mM. Pegylated catalase (pegCAT; cat # C4963; Sigma-Aldrich) was used at a concentration of 500 U/ml. BA receptor antagonists (NF449 [95 (link)], CINPA1 [96 (link)], DY268 [97 (link)], and GSK2033 [98 (link)]) were acquired from Tocris Bioscience (Bristol, UK), and ketoconazole [99 (link)] was purchased from Sigma-Aldrich. NF449 (Gsα-selective antagonist; 5 µM) was used to inhibit TGR5 signaling. Nuclear receptor activation was inhibited using 5 µM CINPA1 (CAR antagonist), DY268 (FXR antagonist), and GSK2033 (LXR antagonist). PXR downstream signaling was inhibited using ketoconazole (5 μM). Chemotherapy drugs (irinotecan, paclitaxel, gemcitabine, 5-fluorouracil and oxaliplatin) were purchased from Sigma-Aldrich. Silencer Select siRNAs targeting TGR5 (GPBAR1-siRNA ID: s195791), VDR (NR1I1- siRNA ID: s14777), and FXR (NR1H4-siRNA ID: s19371), and the negative control siRNA #1 (cat # 4390843) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and used at a final concentration of 30 nM.
Nf449
Manufactured by Bio-Techne
Sourced in United Kingdom
NF449 is a laboratory reagent produced by Bio-Techne. It functions as a selective antagonist for the NF-kappa-B p65 subunit.
Automatically generated - may contain errors
Lab products found in correlation
Apyrase, by Merck Group (3 mentions)
Nf110, by Bio-Techne (3 mentions)
Mrs2179, by Bio-Techne (3 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (3 mentions)
Cinpa1, by Bio-Techne (2 mentions)
Dy268, by Bio-Techne (2 mentions)
Gsk2033, by Bio-Techne (2 mentions)
Forskolin, by Merck Group (2 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Gdc 0449, by Selleck Chemicals (2 mentions)
Trypsin dnase, by Worthington (2 mentions)
Lx 270 426, by Merck Group (2 mentions)
Rolipram, by R&D Systems (2 mentions)
Percoll, by Merck Group (2 mentions)
Nf023, by Bio-Techne (2 mentions)
5 bdbd, by Bio-Techne (2 mentions)
Sq22536, by Bio-Techne (2 mentions)
Forskolin, by Bio-Techne (2 mentions)
Isoproterenol, by Bio-Techne (2 mentions)
26 protocols using nf449
1
Modulation of Cellular Pathways by Bile Acids
2
Modulation of C. parvum Interaction with Bovine PMN
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Bovine PMN were pre-treated with the mentioned inhibitors for 30 min and then co-cultured with C. parvum sporozoites (1:3 PMN/sporozoite ratio, 3 h, 37 °C) with the purpose of blocking the ATP purinergic receptor P2X1, MCT1, MCT2, and glycolysis under hyperoxia 21% O2 = oxygen conditions widely used in most C. parvum-related studies) and physioxia (5% O2) conditions, simulating physiological oxygen pressure of small intestine in vivo as follows [4 (link),12 (link),54 (link),59 (link)]: AR-C 155858 (1 μM, MCT1- and MCT2-inhibitors, Tocris Bioscience, Wiesbaden, Germany), AR-C 141990 (1 μM, MCT1 inhibitor, Tocris Bioscience, Wiesbaden, Germany), NF449 (100 μM, purinergic receptor P2X1 antagonist, Tocris Bioscience, Wiesbaden, Germany), oligomycin A (5 μM, inhibitor of mitochondrial respiration, Sigma-Aldrich, Darmstadt, Germany) and 2-DG (2 mM; antagonist of glycolysis, Sigma-Aldrich, Darmstadt, Germany), were used in this study [37 (link),44 (link),60 (link),61 (link)].
3
Detailed Sourcing of Pharmacological Agents
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BAA was purchased from Zelang Bio-Pharmaceuticals (Nanjing, China) and NF449 was from Tocris Bioscience (Bristol, UK). 2′,5′-dideoxyadenosine (DDA) and N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H-89) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while KG-501 and SP600125 were from Sigma-Aldrich (St. Louis, MO, USA) and SB203580 and U0126 were from Selleck Chemicals (Houston, TX, USA). Levo-tetrahydropalmatine was a gift from Dr. Yan Zhang at Shanghai Jiao University School of Pharmacy. KG-501, SB203580, U0126, and SP600125 were dissolved in dimethyl sulfoxide (DMSO) and made 20% stock solution in saline, while other drugs and reagents were dissolved in 0.9% normal saline.
4
Isolation and Culture of Cerebellar Granule Neurons
5
Antagonists of P2 Purinergic Receptors
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P2 receptor antagonists: Pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), MRS2211, MRS2179, MRS2159, A438079, Brilliant Blue G, ATP-2′, 3′-dialdehyde (OxATP), niflumix acid, carbenoxolone disodium salt, and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, United States). NF449, NF023, and Evans Blue tetrasodium salt were purchased from Tocris Bioscience (Abingdon, United Kingdom). Suramin hexasodium salt was purchased from Aladdin (Shanghai, China). Anti-His monoclonal antibody was purchased from EARTHOX Life Sciences (Millbrae, CA, United States). Anti-ETX monoclonal antibody was developed previously by colleagues in our laboratory. HRP-coupled goat anti-mouse IgG antibody was purchased from TransGen Biotech (Beijing, China). The ATP assay kit was purchased from Calbiochem-Merck (Darmstadt, Germany). MTS (3-(4, 5-dimethylthiazol-2-yl) -5(3-carboxymethoxyphenyl) -2-(4-sulfopheny)-2H-tetrazolium, inner salt) was purchased from Promega Corporation (Madison, WI, United States).
6
P2X1 inhibition in bovine PMN-Cryptosporidium interaction
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For P2X1-inhibition assays, freshly isolated neonatal bovine PMN were pre-treated with NF449 (10 μM, Tocris; purinergic receptor antagonist for P2X1) for 30 min and then co-cultured with C. parvum oocysts (1:2 PMN/oocyst ratio, 3 h, 37°C, 5% CO2). The inhibitor concentration was selected based on previous NET-related studies (15 (link), 40 (link)).
7
Specific P2X1 Receptor Antagonist NF449 Protocol
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NF449 (Tocris Bioscience, Bristol, UK) was used as a specific P2X1 receptor antagonist42 (link) at a concentration of 60 μM, unless otherwise stated. NF449 did not bind to Stx1 or Stx2 in the fluid phase, nor did it affect Stx binding to cells, its uptake or intracellular cleavage as further described in the supplement and Supplementary Fig. S8 (binding) and S9 (uptake).
8
Suramin and Fondaparinux Sodium Protocol
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Suramin hexasodium salt, NF023, NF110, NF157, NF279, NF340, NF449, PSB0739, and MRS2578 were purchased from Tocris Bioscience. Fondaparinux sodium (Arixtra) was purchased from GlaxoSmithKline. Pirodavir was a kind gift from Dr. John Lambert, Biota Pharmaceuticals. MRS2578 and Pirodavir were dissolved in DMSO and used for the experiments.
9
Mast Cell Signaling Pathway Analysis
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UTP, ATP, ADP, PGE1, PGE2, 2,4-dinitrophenyl human serum albumin (DNP-HSA), anti-DNP IgE (clone SPE-7), p-nitrophenyl N-acetyl-β-d-glucosaminide, and the GenElute Mammalian Total RNA miniprep kit were obtained from Sigma-Aldrich (Tokyo, Japan). Allophycocyanin (APC)-conjugated rat anti-mouse c-Kit antibodies (clone 2B8) were obtained from BD Pharmingen (Tokyo, Japan). Phycoerythrin (PE)-conjugated mouse anti-mouse FcεRIα antibodies (clone MAR-1) were obtained from eBioscience (San Diego, CA, USA). Recombinant mouse interleukin (IL)-3 and recombinant mouse SCF were obtained from Peprotech (London, UK). MRS2179, AR-C118925, NF449, AZ10606120, 5-BDBD, Ivermectin, PP2, LY303511, LY294002, Triciribine, and AS605240 were obtained from Tocris Bioscience (Bristol, UK). R406 was obtained from Cayman Chemical (Michigan, USA). Fura-2-acetoxymethylester (AM) and PTX were obtained from Wako (Osaka, Japan). Anti-phospho-Syk, anti-Syk, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, and anti-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). ONO-DI-004 (selective EP1 agonist), ONO-AE1-259-01 (selective EP2 agonist), ONO-AE-248 (selective EP3 agonist), and ONO-AE1-329 (selective EP4 agonist) were obtained from ONO Pharmaceuticals (Osaka, Japan). All other chemicals were of reagent-grade or the highest quality available.
10
Isolation and Culture of Cerebellar Granule Neurons
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Cerebella from P6 or P7 mice were digested with Trypsin/DNase (1 mg ml–1; Worthington), triturated to obtain a single-cell suspension, and then centrifuged through a 35%–65% Percoll gradient (Sigma) according to Yang et al., 2008 13 (link). Cells from the 35%–65% interface were suspended in the GNP culture medium [Neurobasal (Gibco) with 2 mM L-glutamine, 0.45% D-glucose, B27 supplement, 16 μg ml–1 N-Acetyl-LCysteine and penicillin/streptomycin]. We pre-plated GNPs onto poly-D-lysine (100 μg ml–1) coated plates for 1 hr at 37°C twice, and then transferred them to Poly-D/L-ornithine coated plates for culture. We treated GNP cells from control and Gsα mutants with following agents: SAG (Enzo, Lx-270-426) 200 nM; forskolin (Sigma, F3917) 10 μM; a selective Gαs antagonist NF449 (http://www.tocris.com/dispprod.php?ItemId=1801#.UwpK4v3j7Ko ) (Tocris, Cat. # 1391) 200 μM; GDC-0449 (Selleckchem, S1082) 1 μM and Rolipram 50 μM (R&D, Cat.# 0905) or transfected the cells with pcDNA3 or pGsCA for 48 hr. For in vitro proliferation assays, we labeled GNP cells with BrdU (50 μg ml–1) for 48 hr followed by immunostaining.
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