Pepinh trif

Manufactured by InvivoGen

Sourced in United States

Pepinh-TRIF is a laboratory reagent that acts as an agonist of the TRIF (TIR-domain-containing adapter-inducing interferon-β) signaling pathway. TRIF is an adaptor protein involved in the activation of innate immune responses triggered by Toll-like receptor (TLR) 3 and TLR4 signaling. Pepinh-TRIF is a synthetic peptide that mimics the TRIF protein and can be used to stimulate the TRIF-dependent signaling cascade in cell-based assays.

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Lab products found in correlation

Pam3csk4, by InvivoGen (2 mentions) Pepinh myd, by InvivoGen (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Poly 1 c, by Merck Group (2 mentions) Pepinh myd88, by InvivoGen (2 mentions) Lps eb ultrapure, by InvivoGen (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Hyclone, by Thermo Fisher Scientific (1 mentions) Poly 1 c, by InvivoGen (1 mentions) Epoxomicin, by Merck Group (1 mentions) G 6976, by Merck Group (1 mentions) Ku55933, by Merck Group (1 mentions) Panepoxydone, by Enzo Life Sciences (1 mentions) Rupintrivir, by Santa Cruz Biotechnology (1 mentions) Bafilomycin a1, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor, by Merck Group (1 mentions) Butyric acid, by Merck Group (1 mentions) Colchicine, by Merck Group (1 mentions) Draq5, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Carl Roth (1 mentions)

9 protocols using pepinh trif

TLR Ligand Stimulation of PBMCs

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PBMCs were seeded at 1×10⁶ cells/ml in a 24-well plate and stimulated with the following TLR ligands at the concentration of 1 µg/ml Pam3CSK4 (TLR2), 10 µg/ml polyIC (TLR3), 0.5 µg/ml LPS-EB ultrapure (TLR4), 1 µg/ml CLO75 (TLR7/8), 4 µg/ml CpG (ODN 2006) (TLR9) (all Invivogen, San Diego, CA, USA) or PBS (HyClone, Thermo Scientific) for 24 hours.
For inhibition of TLR3 signalling, PBMCs were pre-treated for 6 hours with 5 µM PepinhTRIF (Invivogen) and subsequently stimulated with polyIC as described above.

Kiene M., Rethi B., Jansson M., Dillon S., Lee E., Lantto R., Wilson C., Pöhlmann S, & Chiodi F. (2014). Toll-Like Receptor 3 Signalling Up-Regulates Expression of the HIV Co-Receptor G-Protein Coupled Receptor 15 on Human CD4+ T Cells. PLoS ONE, 9(2), e88195.

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Inhibition of Innate Immune Signaling

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Pepinh-MyD and Pepinh-TRIF (Invivogen) were used alongside the supplied control peptide at 50μM. 1μM 5Z-7-Oxozaeanol (Sigma), 2μg/ml panepoxydone (Enzo Life Sciences), 200nM IKK Inhibitor VII (Millipore), 1μM Bafilomycin A1 (Santa Cruz Biotechnology), 20nM CID1067700 (Millipore), Epoxomicin (Millipore) was added at 2μM, KU55933 (Millipore) was used at 10μM, Gö6976 (Millipore) at 5μM, DBeQ (BioVision) at 8μM and rupintrivir (Santa Cruz Biotechnology) was used at 2μM, were all added 1h prior to infection.

Tam J.C., Bidgood S.R., McEwan W.A, & James L.C. (2014). Intracellular sensing of complement C3 activates cell autonomous immunity. Science (New York, N.Y.), 345(6201), 1256070.

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Inflammatory Signaling Pathway Modulators

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LPS (Eb-ultrapure, 0111:B4), Pam3CSK4, pepinhTRIF, and TAK-242 were obtained from InvivoGen, nigericin was obtained from Invitrogen, and Bacillus anthracis PA was obtained from List Biological Laboratories. Colchicine was obtained from Sigma, and VX-765 was obtained from Sellekchem. BAA-473 was a gift from Dr. Canham (Novartis). DRAQ5 was purchased from eBioscience. TNFα, IFN-β, IFN-γ, M-CSF, IL-3, IL-4, and IL-10 were purchased from Immunotools. TcdA and TcdB from C. difficile strain VPI10463 were recombinantly produced and supplied by Prof. Ralf Gerhard [40 (link)]. Both toxins are identical to TcdA and TcdB from strain cdi630, which was used for infection assay. The HTRF kits for human IL-1β and TNFα were obtained from Cisbio; the ELISA kit for human and mouse IL-1β was obtained from R&D Systems. Both were used according to the manufacturer’s instructions. For the C. difficile supernatant transfer, the following reagents were used: Butyric acid (Sigma-Aldrich: W222119-1KG-K), Various amino acids (Roth or Sigma), Iron sulfate heptahydrate (Sigma-Aldrich: 215422-250G), Triton-X 100 (Roth: 3051.2), M-Per (Sigma-Aldrich: 78501), Protease inhibitor (Sigma-Aldrich: 11836170001).

Mangan M.S., Gorki F., Krause K., Heinz A., Pankow A., Ebert T., Jahn D., Hiller K., Hornung V., Maurer M., Schmidt F.I., Gerhard R, & Latz E. (2022). Transcriptional licensing is required for Pyrin inflammasome activation in human macrophages and bypassed by mutations causing familial Mediterranean fever. PLOS Biology, 20(11), e3001351.

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Investigating Glutamate Metabolism in Neuroinflammation

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Lysozyme (Sigma #L6394), LPS (Sigma #L2630), Glutamate assay kit (Sigma #MAK004) and Minocycline were purchased from Sigma. TLR4 (Abcam #ab22048), PGP9.5 (Abcam #ab108986), GOT (Abcam #ab221939) and Glutaminase (Abcam #ab156876) antibodies were purchased from Abcam. NFκB p65 (CST #8242), Glutamate dehydrogenase (CST #80063), GOT1 (CST #34423), and GOT2 (CST #71692) antibodies were purchased from Cell Signaling Technology. PEPinh MyD, PEPinh TRIF were purchased from Invivogen. Cu-CPT22 (#4884), TAK-242 (#6587), and Dynasore (#2897) were purchased from Tocris. TRIF siRNA (Thermo, #4457308), GOT2 siRNA (Thermo, #4457308), HMGB1 (Thermo #34-8401-82), IFNα mouse ELISA kit (Thermo #BMS6027) and IL-10 mouse ELISA kit (Thermo #BMS614) were purchased from ThermoFisher Scientific. IFN-β ELISA kit (PBL Assay Science #424001) was purchased from PBL Assay Science and mouse inflammatory cytokines ELISA array kit (Qiagen #336161) was purchased from Qiagen. Cell culture media and reagents were purchased from Thermo (Invitrogen). All other reagents were purchased from Sigma until stated otherwise.

Yadav S., Singh A., Kant R, & Surolia A. (2023). TLR4 activation by lysozyme induces pain without inflammation. Frontiers in Immunology, 14, 1065226.

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Modulating Leishmania Infection through siRNA and Inhibitors

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1 μg of appropriate siRNA or control siRNA were added to the RAW264.7 cells to achieve transfection of the desired level, following manufacturer's instructions (Santa Cruz Biotechnology; sc-40261, sc-40257 & sc-45987 for TLR4 siRNA, TLR2 siRNA & MyD88 siRNA, respectively). Briefly, 100 μl siRNA Transfection Medium (prepared as per manufacturer’s protocol) has been added to RAW264.7 (10⁶) cells, for 6 h. Then the cells are washed to remove the medium and been infected with Leishmania parasites followed by treatment with Q-gal or K-gal. Pepinh-TRIF (TRIF Peptide Inhibitory; Cat Code: tlrl-pitrif) brought from InvivoGen. Briefly, the assay with Pepinh-TRIF involves platting of RAW264.7 cells (10⁶) in presence or absence of Pepinh-TRIF (100 μM), for 6 h, followed by infection and treatment with Leishmania parasites and Q-gal/K-gal respectively.

Pradhan S., S.n.e.h.l.a.t.a., Manna D., Karmakar S., Singh M.K., Bhattacharya A., Mukherjee B, & Paul J. (2022). Activation of TLR-pathway to induce host Th1 immune response against visceral leishmaniasis: Involvement of galactosylated-flavonoids. Heliyon, 8(7), e09868.

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Investigating Inflammatory Responses to TLR Agonists

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Damaged and not-damaged cultures (controls) were stimulated with the TLR agonists poly(I:C) (Sigma-Aldrich) (0.1 µg/mL, 1 µg/mL, 10 µg/mL) and LPS (Sigma-Aldrich) (1 µg/mL, 10 µg/mL, 50 µg/mL, 100 µg/mL) for 60 min; the allergen Der p1 (Indoor Biotechnologies Ltd., Cardiff, UK); and conditioned media from virus-infected epithelial cells. TLR agonists and conditioned media were added either alone or in combination with the TLR inhibitor LPS-RS (Sigma-Aldrich) or the TLR inhibitor chloroquine (Sigma-Aldrich) or the intracellular pathway inhibitor Pepinh-TRIF (InvivoGen, Toulouse, France) or the intracellular pathway inhibitor Pepinh-MyD88 (InvivoGen).

Lewandowska-Polak A., Brauncajs M., Jarzębska M., Pawełczyk M., Kurowski M., Chałubiński M., Makowska J, & Kowalski M.L. (2018). Toll-Like Receptor Agonists Modulate Wound Regeneration in Airway Epithelial Cells. International Journal of Molecular Sciences, 19(8), 2456.

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Lymphoma Cell Culture Protocols

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The MDCC-MSB1 cell line, a Marek’s disease lymphoma-derived chicken cell line, was cultured at 38.5 °C in 5% CO₂ in RPMI 1640 medium (Gibco, USA) containing 10% fetal calf serum (Gibco, Australia) and 10% tryptose phosphate broth (Sigma-Aldrich, USA). Avian leukosis virus (ALV) lymphoma-derived chicken cell line DT40 was cultured at 38.5 °C in 5% CO₂ in RPMI 1640 medium (Gibco, USA) containing 10% fetal calf serum (Gibco, Australia), 5% chicken serum (Gibco, USA) and 5% tryptose phosphate broth (Sigma-Aldrich, USA). Poly (I:C) was obtained from Sigma-Aldrich (USA). The pan-caspase inhibitor Z-VAD-FMK was purchase from Beyotime Biotechnology (Nantong, China). The inhibitors Pepinh-TRIF, resveratrol, BX795, and 2-aminopurine were purchase from InvivoGen (USA). Necrostatin-1 and BAY11-7082 was purchased from Abcam (USA).

Zou H., Su R., Ruan J., Shao H., Qian K., Ye J., Yao Y., Nair V, & Qin A. (2017). Double-stranded RNA induces chicken T-cell lymphoma apoptosis by TRIF and NF-κB. Scientific Reports, 7, 7547.

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Macrophage Immune Signaling Modulation

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Toll like receptor adaptor molecule 1(TICAM1 or TRIF) and MYD88 innate immune signal transduction adaptor (MYD88) of macrophages were inactivated by incubating with Pepinh-TRIF (5 µM; Invivogen, San Diego, CA, USA) and Pepinh-MyD88 (5 µM; Invivogen), respectively, for 6 h at 37 °C before the cells were subjected to stimuli. FcγRs of macrophages were blocked by incubation with anti-FcγRI (CD64; BD Biosciences, San Diego, CA, USA), anti-FcγRIIa (CD32a; Stem Cell Technologies, Vancouver, Canada), anti-FcγRIIb (CD32b; OmnimAbs, Alhambra, CA, USA), anti-FcγRIII (CD16; BD Biosciences) antibodies, or mouse IgG (Sigma-Aldrich) at 20 mg/ml for 30 min at 37 °C, after which the cells were subjected to stimuli, resulting in a final antibody concentration of 5 mg/ml.

Chen Q., Wang J., Zhang Q., Zhang J., Lou Y., Yang J., Chen Y., Wei T., Zhang J., Fu Q., Ye M., Zhang X., Dang X., Liang T, & Bai X. (2019). Tumour cell-derived debris and IgG synergistically promote metastasis of pancreatic cancer by inducing inflammation via tumour-associated macrophages. British Journal of Cancer, 121(9), 786-795.

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Investigating TLR3's Role in Cytokine-Induced IFN-γ

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To examine the indispensable role of TLR3 on IL-2 þ IL-12-induced IFN-c production, KHYG-1 cells were treated with a TRIF inhibitor. KHYG-1 cells were seeded into 24-well plates (5 Â 10 5 cells/well) and incubated with 12.5 mg/mL poly(I:C) and 50 mM of the TRIF inhibitor peptide (Pepinh-TRIF, InvivoGen, San Diego, CA) for 24 h under presence or absence of IL-2 and IL-12. The optimal concentration of Pepinh-TRIF was suggested by the manufacturer and we confirmed Pepinh-TRIF had no cytotoxic effect on KHYG-1 cells by preliminary experiment (data not shown). The culture supernatants were collected and centrifuged at 3000g at 4 C for 5 min to remove cellular debris; samples were stored at À80 C until use.

Majima-Horiuchi H., Komine-Aizawa S., Karasaki-Suzuki M., Izumi Y., Aizawa S, & Hayakawa S. (2018). Synergistic induction of interferon-γ by interleukin-2, interleukin-12 and poly(I:C) in a human natural killer cell line. Immunological medicine, 41(3).

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