Recombinant human tnf α 300 01a

Manufactured by Thermo Fisher Scientific

Sourced in United States

Recombinant human TNF-α (300-01A) is a lab equipment product manufactured by Thermo Fisher Scientific. It is a recombinant form of the human tumor necrosis factor-alpha (TNF-α) protein.

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TNF-α (2204 citations) Recombinant human TNF-α (222 citations) Human TNF-α (85 citations) Recombinant TNF-α (45 citations) Recombinant human TNF (28 citations) TNF-α (300-01A) (12 citations) Recombinant human TNF (300-01A; (3 citations)

8 protocols using recombinant human tnf α 300 01a

Recombinant Protein and Bacterial Component Treatments

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Recombinant human SAA1 (hu rSAA1; 300-53), recombinant human CXCL8 (200-08M), recombinant human IL-1β (200-01B) and recombinant human TNF-α (300-01A) were purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant murine SAA3 (mu rSAA3) was purchased from either MyBioSource (MBS1043052; San Diego, CA, USA) or Gentaur (CSB-EP361411Moe1; Kampenhout, Belgium). Lipopolysaccharide (LPS; 0111:B4) derived from E. coli and peptidoglycan (PGN; 77140) derived from Staphylococcus aureus were purchased from Sigma-Aldrich (St. Louis, MO, USA). TAK-242 (HY-11109) was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and was solubilized in DMSO.

Abouelasrar Salama S., De Bondt M., Berghmans N., Gouwy M., de Oliveira V.L., Oliveira S.C., Amaral F.A., Proost P., Van Damme J., Struyf S, & De Buck M. (2020). Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case. Mediators of Inflammation, 2020, 6087109.

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Synthesis and Characterization of IKKβ Inhibitors

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NSC676914A (Additional file 1: Figure S1A) was synthesized by diazotization of 2-[(4-aminophenyl)sulfonyl]-1-hydrogen sulfate followed by coupling with m-toluidine. An unsulfated alcohol analog (Additional file 1: Figure S1B) was similarly prepared from 2-[(4-aminophenyl)sulfonyl]-1-ethanol. Both compounds were purified to >95% purity by reverse phase HPLC. DMSO stocks were used for all experiments. IKKβ inhibitor (IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide, catalog #401484) (Additional file 1: Figure S1C) was purchased from EMD Biosciences (La Jolla, CA). LPA (857130C) was purchased from Avanti Polar Lipids (Alabaster, AL). LPA stocks were made in PBS containing 1% fatty acid-free bovine serum albumin. TPA (4174) was purchased from Cell Signaling Technology, Inc (Danvers, MA). Recombinant Human TNFα (300-01A) was purchased from Peprotech (Rocky Hill, NJ). Z-VAD-FMK (2163) and Necrostatin-1 (2324) were purchased from Tocris Bioscience (Ellisville, MO). Puromycin, XTT, PMS, LPS (L5293) and N-Acetyl-L-cysteine (A7250) were purchased from Sigma-Aldrich (St. Louis, MO).

Sagher E., Hernandez L., Heywood C., Pauly G.T., Young M.R., Schneider J., Colburn N.H, & Annunziata C.M. (2014). The small molecule NSC676914A is cytotoxic and differentially affects NFκB signaling in ovarian cancer cells and HEK293 cells. Cancer Cell International, 14, 75.

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Preparation of Cell Culture Reagents

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All stock solutions were prepared at 1000× concentration. 100 mM caffeine (108160100, ACROS Organics) was prepared as an aqueous solution and was stored at 4 °C for up to 4 weeks. The solution was warmed to 37 °C before experiments to dissolve precipitates. For dose–response experiments, working solutions were prepared by serial dilution in DMEM with 10% (v/v) FBS (F7524, Sigma-Aldrich) and 1% (v/v) streptomycin/penicillin (L0022, Biowest), prewarmed to 37 °C. 10 µg/mL recombinant human TNFα (300-01A, Peprotech),
Aqueous solutions of 10 µg/mL recombinant human FGF-basic (154 a.a.) (100-18B, Peprotech) and 10 µg/mL IL-6 (200−06, PeproTech) were prepared and stored at −20 °C prior to use.
A solution of 10 mM forskolin (F3917, Sigma-Aldrich) in ethanol was prepared and stored at −20 °C prior to use.
An aqueous solution of 4 M KCl (P9541, Sigma-Aldrich) was prepared and stored at 4 °C.
A solution of 10 µM rapamycin (AG-CN2-0025-C100, AdipoGen) in DMSO was prepared and stored in aliquots at −80 °C.

Scheller L., Schmollack M., Bertschi A., Mansouri M., Saxena P, & Fussenegger M. (2020). Phosphoregulated orthogonal signal transduction in mammalian cells. Nature Communications, 11, 3085.

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NF-κB Pathway Activation Assay

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HEK 293T cells and U937 cells were cultured in DMEM and RPMI 1640 medium supplemented with 10% fetal bovine serum, respectively. Streptavidin-Agarose (S1638) and 5-Aza-CdR (A3656) were purchased from Sigma. Recombinant human TNFα (300-01A) was from Peprotech (Changchun, Jilin, China). Antibodies against p65 (sc-372) and p50 (sc-7178) were purchased from Santa Cruz Biotechnology (Changchun, Jilin, China). Goat Anti-Rabbit IgG (111-035-003) was from Jackson ImmunoResearch (Changchun, Jilin, China).

Wang T., Li J., Ding K., Zhang L., Che Q., Sun X., Dai Y., Sun W., Bao M., Wang X., Yang L, & Li Z. (2017). The CpG Dinucleotide Adjacent to a κB Site Affects NF-κB Function through Its Methylation. International Journal of Molecular Sciences, 18(3), 528.

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Synthesis and Characterization of Novel Pyrrolo[3,2-d]pyrimidine Compounds

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Compound 110 (2-methyl-4-(3-methylpiperidin-1-yl)-7-(phenylsulfonyl)-5H-pyrrolo[3,2-d]pyrimidine-6-amine), compound 187 (4-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-7-(2-fluorobenzenesulfonyl)-2-methyl-5H-pyrrolo[3,2-d]pyrimidin-6-amine; Barker et al., 2015 ), and phenylacetyl aminothiazole (PAAT) ((S)-2-(4-chlorophenyl)-N-(5-fluorothizol-2-yl)-3-methylbutanamide; Wang et al., 2010 (link)) were synthesized according to the patent guidelines. Compounds 110 and 187 were completely soluble in dimethyl sulfoxide (DMSO) at a concentration of 20 mM. sodium acetate and forskolin were obtained from Sigma Aldrich (St. Louis, MO, USA), AR420606 (Cat No. 17531) from Cayman Chemical (Ann Arbor, MI, USA), recombinant human TNF-α (300-01A) from PeproTech (Rocky Hill, NJ, USA), and cumate stock (10,000X) from System Biosciences (Palo Alto, CA, USA).

Park B.O., Kang J.S., Paudel S., Park S.G., Park B.C., Han S.B., Kwak Y.S., Kim J.H, & Kim S. (2021). Novel GPR43 Agonists Exert an Anti-Inflammatory Effect in a Colitis Model. Biomolecules & Therapeutics, 30(1), 48-54.

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Pharmacological Modulators of Cellular Pathways

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Actinomycin D (HY-17559), EX527 (HY-1545), MG132 (HY-13259), obeticholic acid (HY-12222), SRT1720 hydrochloride (HY-15145) and tropifexor (HY-107418) were purchased from MedChemExpress. Cycloheximide (HY-12320), nicotinamide (98-92-0) and dimethyl sulfoxide (67-68-5) were purchased from Sigma–Aldrich. DAPI staining solution (C1005), leptomycin B (S1726) and trichostatin A (S1893) were purchased from Beyotime. Recombinant human TNFα (300-01 A) was purchased from Peprotech. Recombinant human FasL (10,244-H07Y) and recombinant human TRAIL (10409-HNAE) were purchased from Sino Biological. Recombinant murine TNFα (14-8321-63) was purchased from Bioworld Technology.

Cui S., Hu H., Chen A., Cui M., Pan X., Zhang P., Wang G., Wang H, & Hao H. (2022). SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR. Acta Pharmaceutica Sinica. B, 13(2), 559-576.

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Signaling Pathway Reagents and Antibodies

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Hank's Balanced Salt Solution (HBSS, 14025092), Opti-Minimal Essential Medium (Opti-MEM; 31985070), and Roswell Park Memorial Institute (RPMI) 1640 medium (11875093) were purchased from Thermo Fisher Scientific (Waltham, MA). Bovine serum albumin (BSA; A9418) was obtained from Sigma-Aldrich (St. Louis, MO), and collagenase IV (LS004188) was sourced from Worthington Biochemical Corporation (Lakewood, NJ). U0126 (S1102), PD98059 (S1177), SP600125 (S1460), and SB203580 (S1076) were purchased from Selleckchem (Houston, TX). Recombinant human TNFα (300-01A) was obtained from PeproTech (Rocky Hill, NJ). Ficoll-Paque PLUS (17-5442-02) was purchased from GE Healthcare (Marlborough, MA). Mouse anti-human MMP3 antibody (Ab; 1B4, sc-21732) and mouse anti-human β-actin (C4, sc-47778) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX). Rabbit antibodies for the following proteins and phosphoproteins were purchased from Cell Signaling Technology (Beverly, MA): phospho-p44/42 mitogen-activated protein kinase (MAPK; T202/Y204) (9101), p44/42 MAPK (Erk1/2) (9102), phospho-SAPK/c-Jun N-terminal kinase (JNK) (T183/Y185) (9251), SAPK/JNK (9252), phospho-p38 MAPK (T180/Y182) (9211), and p38 MAPK (9212).

Pak H.K., Kim Y.W., Nam B., Lee A.N., Roh J., Gil M., Liu C., Chung Y.S, & Park C.S. (2021). The Migration of Human Follicular Dendritic Cell-Like Cell Is Facilitated by Matrix Metalloproteinase 3 Expression That Is Mediated through TNFα-ERK1/2-AP1 Signaling. Journal of Immunology Research, 2021, 8483938.

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Recombinant HCV Proteins and Immune Modulation

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Recombinant HCV Core protein (MBS596029; Sequence ID BAM73001.1), NS3 protein (MBS596033; Sequence ID AIZ00641.1) and glycoprotein E2 (MBS434003; Sequence ID AAR22408.1) derived from genotype 1b were obtained from MyBiosource (San Diego, CA, USA). The lipopolysaccharide (LPS) content of the recombinant HCV proteins was as low as 0.02 ng/100 µg of core protein, 0.007 ng/100 µg of NS3 and 0.7 ng/100 µg of glycoprotein E2.
The maximal level of LPS reached in the highest tested concentrations of the viral proteins did not influence any of the activities reported here (data not shown). LPS (O111:B4) derived from E.coli and dsRNA (P1530) were obtained from Sigma-Aldrich (Overijse, Belgium). Mouse monoclonal IgG2a isotype control (MAB0031) and antibodies against human CCL2 (MAB679, BAF279), CXCL10 (BAF266) and CXCL8 (MAB208) were obtained from R&D Systems (Minneapolis, MN, USA). Recombinant human CCL3 (270-LD) and recombinant human IFN-γ (285-IF) were obtained from R&D Systems. Capture monoclonal anti-human CXCL10 (500-P93), recombinant human IL-1β (200-01B) and recombinant human TNF-α (300-01A) were obtained from Peprotech (London, UK). Mouse monoclonal anti-TLR2 IgG2a antibody (16-9922-82) was obtained from ThermoFischer Scientific (Merelbeke, Belgium). TAK-242 (HY-11109) was obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Abouelasrar Salama S., Gouwy M., De Zutter A., Pörtner N., Vanbrabant L., Berghmans N., De Buck M., Struyf S, & Van Damme J. (2020). Induction of Chemokines by Hepatitis C Virus Proteins: Synergy of the Core Protein with Interleukin-1β and Interferon-γ in Liver Bystander Cells. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 40(4).

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