Recombinant human IFN-α (PBL Assay Science), IFN-ε (EnQuire Bio), IFN-γ (PeproTech), and IFN-λ (PeproTech) were purchased from suppliers, resuspended at 50 ng/µl, and added to cell culture at 50 ng/ml, the optimal concentration for HIV-1 restriction determined empirically in our cell culture system. The following units of each IFN tested were used: 7,850 U/test for IFN-α, 500 U/test for IFN-γ, and 125 U/test for IFN-λ. IFN-ε specific activity was untested by the manufacturer and thus utilized at a mass concentration identical to IFN-α, IFN-γ, and IFN-λ. Inhibitors were purchased from suppliers and resuspended at a concentration of 1 mM and added to culture at a concentration of 1 µM.
Recombinant human ifn α
Manufactured by PBL Assay Science
Sourced in United States
Recombinant human IFN-α is a protein produced using recombinant DNA technology. It serves as a key component in various scientific research and assay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Ifn λ, by Thermo Fisher Scientific (1 mentions)
Ifn λ, by PBL Assay Science (1 mentions)
Ifn γ, by PBL Assay Science (1 mentions)
Nsc87877, by Santa Cruz Biotechnology (1 mentions)
Mg132, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Anti phospho stat2, by Cell Signaling Technology (1 mentions)
Anti phospho stat1, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fluoview ver 3, by Olympus (1 mentions)
Polyinosinic polycytidylic acid, by Merck Group (1 mentions)
Necrosulfonamide, by Merck Group (1 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
Z ietd fmk, by R&D Systems (1 mentions)
Birinapant, by Apexbio (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Caspase 3 and 8 colorimetric assay kits, by Abcam (1 mentions)
Sm 164, by Apexbio (1 mentions)
Azd5582, by Chemietek (1 mentions)
7 protocols using recombinant human ifn α
1
Interferon-Mediated HIV-1 Restriction
2
Reagents and Plasmids for Interferon Signaling
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Flag-USP2a and catalytic inactive Flag-USP2a-C276A mutant were gifts from Dr. Simon S. Wing (McGill University). HA-USP2a and HA-USP2b were gifts from Dr. Chengjiang Gao (Shangdong University, China). Flag-STAT2 was a gift from Dr. Chunfu Zheng (Soochow University, China). 39 human USPs expression plasmids were gifts from Dr. J. Wade Harper (Harvard Medical School, Addgene plasmids). Flag-STAT1 was generated using PCR amplified from pIND-STAT1-V5 from Dr. Steven Johnson (Addgene # 11618). HA-Ub was a gift from Dr. Lingqiang Zhang (State Key Laboratory of Proteomics, China). ISRE-Luc, GAS-Luc and Renilla plasmids were gifts from Dr. Serge Y. Fuchs (University of Pennsylvania). shUSP2a, shKPNA1 and shImportin α plasmids were purchased from GENECHEM (Shanghai, China). All the mutations were generated by QuickChange site-Directed Mutagenesis Kit (Stratagene). All the plasmids were confirmed by DNA sequencing. Recombinant human IFNα, IFNγ and IFNλ were purchased from PBL Interferon Source. IFNα was used at the concentration of 1,000 IU/ml, unless stated otherwise. Cycloheximide, MG132, and other chemicals were purchased from Sigma. NSC87877 was from Santa Cruz (sc-204139).
3
Visualizing PRRSV-Induced STAT Signaling
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Marc-145 cells cultured on coverslips in 24-well plates were infected with PRRSV at a multiplicity of infection (MOI) of 0.5. At 24 h postinfection, the infected cells were mock treated or treated with recombinant human IFN-α (PBL Assay Science) (1,000 U/ml) for 4 h. The cells were then fixed with 4% paraformaldehyde for 15 min and immediately permeabilized using methanol (precooled at −20°C) for 10 min at room temperature (RT). After three washes with PBS, cells were incubated with 5% bovine serum albumin (BSA)–PBS overnight at 4°C and incubated with the primary antibody for 1 h. The antibodies used were as follows: anti-phospho-STAT1 (Cell Signaling Technology, USA), anti-phospho-STAT2 (Cell Signaling Technology, USA), anti-IRF9 (Abclonal, China), and anti-nsp11 MAb. Anti-mouse IgG (H+L) antibody conjugated to Alexa Fluor 594 or anti-rabbit IgG (H+L) antibody conjugated to Alexa Fluor 488 was diluted to 1:500 for use as the secondary antibody, after which the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI; Beyotime, Nantong, China)–PBS for 15 min. Fluorescent images were acquired with a confocal laser scanning microscope (Olympus Fluoview ver. 3.1, Japan).
4
SARS-CoV-2 Induced IFN-α/β Activity in Monocytes
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MDBK cells (ATCC, CCL-22) were cultured in Eagle’s Minimum Essential Medium (EMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 50 µg/ml gentamicin (27 (link)). Vesicular stomatitis virus (VSV Indiana laboratory strain) (V-520-001-522, ATCC, VR-1238) was passaged on Vero cells (ATCC, CCL-81), and viral titer was determined using the plaque assay in Vero cells. MDBK cells bind and respond to human type I IFNs, α and β, but not IFN-gamma (IFN-γ) (27 (link)). Culture supernatants were collected from virus-exposed MDMs on days 1, 3, and 5 following infection with SARS-CoV-2 at MOI of 0.01, then assessed for IFN-α/β activity by protection against the VSV-induced cytopathicity measured in MDBK cells (27 (link)). Polyinosinic-polycytidylic acid (poly(I:C); Sigma-Aldrich, P9582) served as a positive control for IFN induction via TLR3 engagement (28 (link)). Recombinant human IFN-α (PBL Assay Science, 11200-2) was used as assay standard. We also investigated IFN activity upon the challenge of Teflon flask-suspended monocytes with SARS-CoV-2 (MOI=0.01), before and after treatment of cells with 100 µg/ml poly(I:C).
5
Modulating Cell Death Pathways
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The Smac mimetic AZD5582 was obtained from Chemietek (Indianapolis, IN, USA) and Smac mimetics SM164, BV6, and Birinapant (TL32711) were from APExBIO (Houston, TX, USA). Recombinant human IFNα was from PBL Assay Science (Piscataway, NJ, USA) and IFNγ, IFNλ, TNFα, and Annexin V-FITC were from eBioscience (San Diego, CA, USA). Recombinant human TRAIL was from ProSpec TechnoGene (East Brunswick NJ, USA). Polyinosinic–polycytidylic acid (poly(I:C)) was from InvivoGen (San Diego, CA, USA). Necrostatin-1, necrosulfonamide, GSK872, Bay11-7082, JAK kinase inhibitor I, AG-1478, and cisplatin were from EMD Millipore (Billerica, MA, USA). The general caspase peptide inhibitor Z-VAD-FMK and the caspase-8 peptide inhibitor Z-IETD-FMK were from R & D Systems (Minneapolis, MN, USA). Human TNFα neutralizing antibody (#7321) was from Cell Signaling Technology (Beverly, MA, USA). Caspase-3 and -8 colorimetric assay kits were from BioVision (Milpitas, CA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
6
Immune Cell Stimulation Assay
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The following reagents were used to stimulate cells: Recombinant human IFN-α (1,000 U/ml, PBL Assay Science, Piscataway, NJ, USA), mouse IFN-α (1,000 U/ml, PBL Assay Science), CpG-B 1826 (10 µg/ml), CpG-A-2336 (5 µg/ml) (IDT Biotechnologies, Coralville, IA, USA), CpG-A-1585 (1 µg/ml) (Invivogen, San Diego, CA, USA), resiquimod (1 µg/ml) (R848; Invivogen) (23 (link), 25 (link)–27 (link)), bradykinin peptide (10 µM), Lys-des-Arg(9)-bradykinin, which is a kinin breakdown product and a selective bradykinin B1 receptor agonist (10 µM), Arg–Pro–Hyp–Gly–Phe–Ser–Pro–Phe–Arg B2 receptor agonist (10 µM), B1 receptor antagonist ([des-Arg10-HOE140]- DH-1 μg/ml), B2 receptor antagonist (HOE140–H–10 μM) (all bradykinin agonists and antagonists were purchased from Sigma-Aldrich, St. Louis, MO, USA) (28 (link)), recombinant human klk1 (1 µg/ml) (Creative Biomart, Shirley, NY, USA), captopril (20 μM)(Sigma) (29 (link)), and indomethacin (indo) (1 µg/ml) (Sigma) (30 (link)).
7
ZIKV Infection and IFN-α Response
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MDMs and moDCs were uninfected or infected with ZIKVPR (MOI=10.0) for 6 h at 37°C. After 6 h, the cells were subsequently washed with PBS and were untreated or treated with 1000U/mL of recombinant human IFN-α (PBL Assay Science). After 6 h of treatment, the cell lysates were harvested for detecting expression of interferon-stimulated genes (ISGs) by qRT-PCR.
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