Irak 1 4 inhibitor

Manufactured by Merck Group

Sourced in United States

IRAK-1/4 inhibitor is a laboratory research tool used to inhibit the activity of IRAK-1 and IRAK-4 proteins. These proteins play a role in inflammatory and immune signaling pathways. The IRAK-1/4 inhibitor can be used to study the effects of modulating these pathways in cell-based or biochemical assays.

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Lab products found in correlation

5z 7 oxozeaenol, by Merck Group (2 mentions) Mycoalert mycoplasma detection kit, by Lonza (1 mentions) Z ietd fmk, by R&D Systems (1 mentions) Cli 095, by InvivoGen (1 mentions) Z vad fmk, by R&D Systems (1 mentions) Er 27319 maleate, by R&D Systems (1 mentions) Cytochalasin d, by R&D Systems (1 mentions) Glycine, by Merck Group (1 mentions) Apocynin, by R&D Systems (1 mentions) Ps 1145, by Cayman Chemical (1 mentions) Ly294002, by Merck Group (1 mentions) Streptomycin sulfate, by Nacalai Tesque (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Flagellin, by Enzo Life Sciences (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Rpmi 1640 medium, by Nacalai Tesque (1 mentions) Sc 514, by Merck Group (1 mentions) Recombinant il 1β, by Thermo Fisher Scientific (1 mentions) Human cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Recombinant il 18, by R&D Systems (1 mentions)

Close spelling variants of this product

Inhibitor IV (2 citations)

9 protocols using irak 1 4 inhibitor

Cell Lines and Patient-Derived Samples for WM Study

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MM cell line MM.1S and WM-derived cell lines BCWM.1 and MWCL-1 were used in this study. BCWM.1 (2010, kind gift from Dr S.P. Treon, Dana-Farber Cancer Institute Boston, MA) and MM.1S (2010, kind gift from Dr S. Rosen, Northwestern University, Chicago, IL) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 100U/mL of penicillin/streptomycin. MWCL-1 (2010, kind gift from Dr S. Ansell, Mayo Clinic, Rochester, MN) was cultured in Iscove Modified Dulbecco Medium supplemented with 10% fetal bovine serum and 100 U/mL of penicillin/streptomycin. Cells were kept at 37°C, 5% CO₂ in humid atmosphere. Phenotypes were tested using flow cytometry at cell line delivery and mycoplasma testing was done using MycoAlert™ Mycoplasma Detection kit (Lonza, Basel, Switzerland) after vial thawing, every two months.
CD19+ cells from WM patients were isolated as described [6 (link)] and cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 100U/mL of penicillin/streptomycin. Informed consent was obtained prior to research sampling. Lenalidomide and pomalidomide were provided by Celgene. All drugs tested were purchased from Selleck chemicals (Houston, TX) apart from the IRAK 1/4 inhibitor (Merck Millipore, Darmstadt, Germany).

Bertrand E., Jouy N., Manier S., Fouquet G., Guidez S., Boyle E., Noel S., Tomowiak C., Herbaux C., Schraen S., Preudhomme C., Quesnel B., Poulain S, & Leleu X. (2017). Role of IRF4 in resistance to immunomodulatory (IMid) compounds® in Waldenström’s macroglobulinemia. Oncotarget, 8(68), 112917-112927.

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Inhibition of Immune Pathways

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Syk inhibitor (ER 27319 maleate), TLR2 inhibitor (human TLR2 mAb, clone 383936), isotype control for TLR2 mAb (mouse IgG2B, clone 20116), pan-caspase inhibitor (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), NADPH-oxidase inhibitor (apocynin) and phagocytosis inhibitor (cytochalasin D) were purchased from R&D Systems. TLR4 inhibitor (CLI-095) was from Invivogen, IRAK-1/4 inhibitor from EMD Millipore, glycine from Sigma-Aldrich. All inhibitors were used at concentrations indicated in the figure legends. All reagents were reconstituted in sterile dimethyl sulfoxide except for the antibodies that were dissolved in PBS and glycine that was dissolved in distilled water.

Tóth A., Zajta E., Csonka K., Vágvölgyi C., Netea M.G, & Gácser A. (2017). Specific pathways mediating inflammasome activation by Candida parapsilosis. Scientific Reports, 7, 43129.

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Inhibition of IL-17-induced IL-6 in Synovial Fibroblasts

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Synovial fibroblasts were plated at a density of 5 × 10⁴ cells per well in 24-well plates with Ham's F12 medium containing 10% FBS. Confluent cells were cultured for 24 h in medium containing 2% FBS. The inhibition experiments were performed using the following inhibitors: Interleukin-1 Receptor-Associated-Kinase-1/4 (IRAK-1/4) inhibitor (20 μM) (Merck KGaA, Darmstadt, Germany), the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (20 μM) (Merck KGaA), the transforming growth factor-β-activated kinase 1 (TAK1) inhibitor (5z)-7-Oxozeaenol (1 μM) (Merck KGaA), and the inhibitor of the NFκB kinase β subunit (IKKβ) inhibitor PS-1145 (10 μL) (Cayman Chemical, Ann Arbor, MI, USA). The cells were pretreated with the inhibitor reagents for 30 min, followed by incubation with IL-17 (10 ng/mL). After 8 h, the culture supernatants were collected and stored at −80°C until use. The inhibitor effect was calculated as 100 − [(IL-6 production with IL-17 in the presence of the inhibitor)/(IL-6 production with IL-17) × 100]. The IL-6 levels in the conditioned medium were measured using ELISA kit (R&D systems).

Hattori T., Ogura N., Akutsu M., Kawashima M., Watanabe S., Ito K, & Kondoh T. (2015). Gene Expression Profiling of IL-17A-Treated Synovial Fibroblasts from the Human Temporomandibular Joint. Mediators of Inflammation, 2015, 436067.

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Gastric Cancer Cell Lines Maintenance

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MKN45cl85 and 85As2 cell lines were established from the human gastric MKN-45 cancer cell line as described previously [7 (link)]. Cells were maintained in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 IU/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate (Nacalai Tesque, Inc.) in a 5% CO₂ atmosphere at 37°C. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Nacalai Tesque, Inc. LPS was purchased from Sigma Chemical (St. Louis, MO, USA). Flagellin was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). IRAK-1/4 inhibitor was purchased from Merck Millipore (Billerica, MA, USA).

Terawaki K., Kashiwase Y., Uzu M., Nonaka M., Sawada Y., Miyano K., Higami Y., Yanagihara K., Yamamoto M, & Uezono Y. (2018). Leukemia inhibitory factor via the Toll-like receptor 5 signaling pathway involves aggravation of cachexia induced by human gastric cancer-derived 85As2 cells in rats. Oncotarget, 9(78), 34748-34764.

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Investigating Adipogenesis Regulation

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After pre-treating the cells with the selective inhibitors; either SC-514 (50 µM, Sigma-Aldrich), IRAK 1/4 inhibitor (10 µM, Sigma-Aldrich), SYK inhibitor (10 µM, Abcam), or IRAK 1/4 plus SYK inhibitors (10 µM) in the growth medium for 30 min, the medium was removed. Then, a fresh D/A medium containing the same concentration of those inhibitors and LCB (1:150 cells:LCB) was added. Cells were harvested at the indicated time points for analyzing the molecular mechanisms of LCB on adipogenesis, as described above.

Jakkawanpitak C., Inafuku M., Oku H., Hutadilok-Towatana N., Bunkrongcheap R., Sermwittayawong N., Aiemchareon P, & Sermwittayawong D. (2021). Mechanism of the fungal-like particles in the inhibition of adipogenesis in 3T3-L1 adipocytes. Scientific Reports, 11, 18869.

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Modulating CD4+ T Cell Polarization

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Peripheral CD4⁺T cells were purified from PBMCs by magnetic-assisted cell sorting (MACS) using a human CD4⁺T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotec, Palo Alto, CA). The purity of isolated CD4⁺T cells, identified by FCM, was more than 95%. IRAK-1/4 inhibitor was obtained from Sigma-Aldrich (St. Louis, MO; catalog number: I5409) and was dissolved in dimethyl sulfoxide (DMSO) at 50 μM as a stock solution and stored at −20°C. CD4⁺T cells were resuspended at 1×10⁶ cells/ml in medium RPMI 1640 (Gibco, Invitrogen, Carlsbad, CA) containing L-glutamine (2 mM), penicillin/streptomycin (100 U/ml), and 10% fetal calf serum. CD4⁺T cells, activated with anti-CD3 (0.5 μg/ml) and anti-CD28 antibodies (0.1 μg/ml), were cultured with IRAK inhibitor (50 nM Sigma) or DMSO in the presence of recombinant IL-18 (100 ng/ml; R&D Systems, Minneapolis, MN) or recombinant IL-1β (20 ng/ml; PeproTech, London, U.K.) for 3 days. IL-18 was used to polarize the cells towards a Th1 subset [18] (link), [19] (link) whereas IL-1β was used to polarize the cells towards a Th17 subset [20] (link).

Sun M., Yang P., Du L., Yang Y, & Ye J. (2014). The Role of Interleukin-1 Receptor-Associated Kinases in Vogt-Koyanagi-Harada Disease. PLoS ONE, 9(4), e93214.

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Gemcitabine and IL-1β Inhibition

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Gemcitabine was purchased from the Siteman Cancer Center Pharmacy. Details of inhibitors were: IRAK1/4 inhibitor (Sigma, cat# I5409), AS2444697 (Tocris, cat#5430), IMD-0354 (Tocris, cat#2611), anti-mouse IL-1β neutralizing antibody (Invivogen, clone 7E3), anti-human IL-1β neutralizing antibody (Invivogen, clone 4H5).

Zhang D., Li L., Jiang H., Li Q., Wang-Gillam A., Yu J., Head R., Liu J., Ruzinova M.B, & Lim K.H. (2018). Tumor-Stroma IL-1β-IRAK4 Feedforward Circuitry Drives Tumor Fibrosis, Chemoresistance, And Poor Prognosis In Pancreatic Cancer. Cancer research, 78(7), 1700-1712.

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Inhibiting MAPK and NF-kB Signaling

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Various inhibitors of mitogen-activated kinases (MAPK), AP-1/CREB were used to block signaling due to TLRs. TRAF-6 inhibitor peptide set and NF-kB inhibitor peptide set (#2004) were from Imgenex, USA, and IRAK1/4 inhibitor (#15409) was from Sigma (USA). AP-1 inhibitor (#SR11302) was from Tocris (USA). MΦs were treated with inhibitors for 2 h, activated with TLR-ligands for 2 h, and infected with BCG for 2 h prior to fixation and assay of surface MHC-II or antigen presentation. This rapid procedure was adapted to ensure >90% viability of MΦs.

Khan A., Bakhru P., Saikolappan S., Das K., Soudani E., Singh C.R., Estrella J.L., Zhang D., Pasare C., Ma Y., Sun J., Wang J., Hunter R.L., Tony Eissa N., Dhandayuthapani S, & Jagannath C. (2019). An autophagy-inducing and TLR-2 activating BCG vaccine induces a robust protection against tuberculosis in mice. NPJ Vaccines, 4, 34.

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Pharmacological Inhibitors Modulating TLR3/dsRNA Signaling

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The IRAK-1/4 inhibitor (#I5409) and the MKK7 inhibitor 5Z-7-Oxozeaenol (#O9890) were purchased from Sigma (St. Louis, MO), and all other inhibitors were purchased from Calbiochem-EMD Millipore (Danvers, MA). For all experiments using pharmacological inhibitors, cells were treated with the inhibitor for 30 min, followed by treatment with PIC for up to 48 h in the presence of the inhibitor. Controls included untreated cells and cells treated only with inhibitor (without PIC). The concentrations of each inhibitor were based on their manufacturer recommended IC₅₀. The TLR3/dsRNA Complex Inhibitor (TLR3.CI, EMD#614310), a thiophenecarboxamidopropionate compound that acts as a direct, competitive and high affinity inhibitor of dsRNA binding to TLR3, was used at concentrations of 5, 10, 20, 30, 40, 50, and 100 μM; the NFκB inhibitor (EMD#481406) at 20 nM and 30 nM, the AP-1 inhibitor (EMD#SR11302) at 2 μM and 10 μM, JNK inhibitor II (EMD#420119) at 10 μM and 20 μM, JNK Inhibitor III (EMD#420130) at 5 μM and 10 μM, JNK inhibitor V (EMD#420129) at 10 μM and 20 μM. The IRAK-1/4 and MKK7 inhibitors were used at 5 μM and 10 μM. The manufacturer’s names, catalog numbers, and mechanisms of action of each inhibitor are summarized in Table-1.

Bhargavan B., Woollard S.M, & Kanmogne G.D. (2015). Toll-Like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication. Cellular signalling, 28(2), 7-22.

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