LPS purified from Escherichia coli O111:B4, TNBS, t-BHP, collagenase type VIII, RPMI 1640 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies for COX-2, ERK, p-ERK, IκBα, p- IκBα, IRAK1, p-IRAK1, iNOS, p65, p-p65, TAK1 and p-TAK1 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Radioimmuno-precipitation assay (RIPA) buffer, tetramethyl benzidine was purchased from Sigma (St Louis, MO, U.S.A.). ELISA kits for IL-1β, IL-6, IL-10, and TNF-α were purchased from R&D Systems (Minneapolis, MN, U.S.A.). mRNA isolation kit was purchased from Qiagen (Hilden, Germany). A diazo-coupled limulus amoebocyte lysate (LAL) assay kit was purchased from Cape Cod Inc. (E. Falmouth, MA, USA). Pan T Cell Isolation Kit II was purchased from MiltenyiBiotec GmbH (Bergisch Gladbach, Germany). Anti-CD28, anti-CD3, recombinant IL-6, and recombinant TGF-β were purchased from BioGems International Inc. (Westlake Village, CA, U.S.A.). Fetal bovine serum (FBS) and heat-inactivated fetal calf serum (FCS) purchased from Panbiotech GmbH (Aidenbach, Germany). de Man, Rogosa and Sharpe (MRS) medium for probiotics was purchased from BD (Sparks, MD, USA). General anaerobic medium (GAM) for probiotics and other bacteria were purchased from Nissui Pharmaceutical Co (Tokyo, Japan). Other chemicals used were of the highest grade available.
Irak1
Manufactured by Cell Signaling Technology
Sourced in United States
IRAK1 is a serine/threonine protein kinase that plays a central role in the interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling pathways. IRAK1 is involved in the activation of NF-kB and MAPK signaling cascades, leading to the production of pro-inflammatory cytokines.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (8 mentions)
Irak4, by Cell Signaling Technology (5 mentions)
P tak1, by Cell Signaling Technology (4 mentions)
P iκbα, by Cell Signaling Technology (3 mentions)
Mek1 2, by Cell Signaling Technology (3 mentions)
Il 1β, by Cell Signaling Technology (3 mentions)
Myd88, by Cell Signaling Technology (3 mentions)
Traf6, by Cell Signaling Technology (3 mentions)
P erk, by Cell Signaling Technology (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Nf κb, by Cell Signaling Technology (2 mentions)
Cox 2, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
Lipopolysaccharides (lps), by InvivoGen (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
24 protocols using irak1
1
Inflammatory Pathway Activation Assay
2
Methanol Extraction of P. attenuatum and Anti-inflammatory Effects
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Methanol extraction of P. attenuatum (Pa-ME) was purchased from the Plant Extract Bank in the Plant Diversity Research Center (Daejeon, Republic of Korea; http://extract.kribb.re.kr , e-mail: mplantext@kribb.re.kr). RAW264.7 cells, a transformed macrophage cell line derived from the BALB/c mouse (ATCC number TIB-71), were purchased from ATCC (Rockville, MD, USA). Dimethyl sulfoxide (DMSO), L-NG-nitroarginine methyl ester (L-NAME), indomethacin, lipopolysaccharide (LPS, Escherichia coli 0111:B4), pam3CSK, and (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Poly I:C was obtained from Calbiochem (La Jolla, CA). The enzyme immune assay (EIA) kits used to quantitate the levels of PGE2 were purchased from Amersham (Little Chalfont, Buckinghamshire, UK). Specific PCR primers for iNOS, TNF-α, COX-2, and GAPDH were synthesized from Bioneer Inc. (Daejeon, Republic of Korea). Antibodies that specify phosphorylated and total forms of p65, p50, c-Jun, c-Fos, Lamin A/C, IκBα, IKKα/β, AKT, Src, Syk, ERK, p38, JNK, IRAK1, IRAK4, TAK1, MKK3/6, and β-actin were obtained from Cell Signaling (Beverly, MA, USA).
3
Immunoblotting Protocol for Inflammatory Signaling
4
Biochemical Characterization of Cell Lines
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HEK293 cells (ATCC number CRL-1573) and RAW264.7 cells (ATCC number TIB-71) were acquired from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Dimethyl sulfoxide (DMSO), carboxymethylcellulose (CMC), 3-(4,5-dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyethylenimine (PEI), sodium dodecyl sulfate (SDS), ranitidine, dexamethasone (dexa), L-NAME, kaempferol, genistin, apigenin, lipopolysaccharide (LPS, E. coli 0111:B4), pam3csk4, and poly (I:C) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). RPMI 1640, DMEM, trypsin, PBS, and penicillin-streptomycin were received from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was acquired from Biotechnics Research, Inc. (Irvine, CA, USA). TRIzol reagent was purchased from MRCgene (Cincinnati, OH, USA). Primers used for semiquantitative RT-PCR and qPCR were purchased from Macrogen Inc. (Seoul, Korea). Phospho-specific or total-protein antibodies against p65, p50, IκBα, PI3K/p85, Src, c-Fos, c-Jun, JNK, ERK, p38, MKK4, MKK7, MEK1/2, TAK1, IRAK1, IRAK4, hemagglutinin (HA), and β-actin were acquired from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Constructs for signaling proteins HA-Src and HA-TAK1 were used as previously reported [24 (link),26 (link)].
5
Proximity Ligation Assay for TLR Activation
6
Protein Expression Analysis in Kidney Samples
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Twenty micrograms of proteins from the kidneys or DC homogenates were separated on 15 or 10% (wt/vol) polyacrylamide denaturing gels and electro-blotted onto polyvinylidene fluoride membranes. The primary antibodies used were anti-caspase-3 (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), IFN-γ (1 : 1000; Cell Signaling Technology), IL-4 (1 : 1000; Cell Signaling Technology), IL-10 (1 : 1000; Cell Signaling Technology), SOCS1-3 (1 : 1000; Cell Signaling Technology), Jak-2 (1 : 1000; Cell Signaling Technology), phospho-STAT3 (1 : 1000; Cell Signaling Technology), NF-κB (1 : 1000; Cell Signaling Technology), TLR-2 (1 : 1000; Cell Signaling Technology), TLR-4 (1 : 1000; Cell Signaling Technology) and IRAK1 (1 : 1000; Cell Signaling Technology). The semiquantitative analysis (AlphaView Software 3.3; ProteinSimple, San Jose, CA, USA) results were expressed as the optical volume densities (OD x mm2) normalized to β-actin (1 : 10 000 dilution; Abcam).
7
Signaling Pathway Protein Analysis
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Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer, centrifuged at 13,000 × g for 10 minutes and supernatants collected. Immunoblots were conducted to determine levels of p-IRF3(ser396), IRF3, p-TBK1/NAK(Ser172), TBK1/NAK, RIG-I, MDA5, LGP2, TLR2, TLR3, TLR7, TLR8, TLR9, Trif, MyD88, IRAK1, NOD2 and actin (all antibodies from Cell Signaling Technologies). TLRs, NOD1, NOD2, NXLR1, RIG-I, MDA5, Trif and actin expressions were determined by qPCR using Taqman probes (Life technologies/Applied Biosystems).
8
Molecular Mechanisms of Anti-Inflammatory Effects
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Dulbecco’s Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), streptomycin and penicillin were obtained from Welgene (Daegu, Republic of Korea). Primers shown in Table 1 were acquired from Bioneer (Daejeon, Republic of Korea). Lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and dextran sulfate sodium (DSS) were from Sigma-Aldrich (Catalogue# 42867, Molecular weight is 40,000 Da, St. Louis, MA, USA). Antibodies for Western blot, iNOS (#2982), COX-2 (#4842), p-TAK1 (#4531), p-NFκB p65 (#3033), IRAK-1 (#4359), p-IKKα/β (#2697), p-IκBα (#2859), β-actin (#4967), T-ERK (#9102), p-ERK (#9101), T-JNK (#9252), p-JNK (#9251), T-p38 (#9212), p-p38 (#9211), NLRP3 (#15101), HRP-linked antibody (#7074) were all obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies conjugated with fluorescence for flow cytometry analysis, PE-Cy5 anti-CD3 (145-2C11; 553065), PE anti-CD4 (RM4-5; 553049), FITC anti-CD8 (53-6.7; 553031), FITC anti-CD25 (3C7; 558689), FITC anti-CD19 (ID3; 553785) and PerCP-Cy5.5 anti-CD69 (H1.2F3; 561931), and CD3e monoclonal antibody (145-2C11; 553058) were obtained from BD Biosciences (San Diego, CA, USA).
9
Western Blot Analysis of Inflammatory Signaling
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Western blot assay was performed as described in our previous study20 (link). Briefly, the protein extracts were heated with the sample buffer for 10 mins, followed by divided on a 10% polyacrylamide gel, and then transferred into the PVDF membrane. The membranes were blocked with 5% BSA, followed by maintained with primary antibodies for MyD88 (1:1000, Cell Signaling Technology), IRAK1 (1:2000, Cell Signaling Technology) and TRAF6 (1:1000, Santa Cruz Biotechnology). In addition, antibodies against IL-1β (1:2000, Santa Cruz Biotechnology), IL-6 (1:1000, Cell Signaling Technology) and TNF-α (1:2000, Santa Cruz Biotechnology) were used. Then the membranes were rinsed in TBST to be incubated with corresponding secondary antibodies at room temperature for 1 h. β-actin was used to act as a loading control. LI-COR Odyssey System was used to vision the protein bands in the membranes.
10
Inflammatory Signaling Pathway Inhibitors
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Polyethylenimine (PEI), PP2, piceatannol (Picea), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (a tetrazole) (MTT), Nω-nitro-L-arginine methyl ester (L-NAME), and lipopolysaccharide (LPS, E. coli 0111:B4) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and RPMI1640 were obtained from GIBCO (Grand Island, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). RAW264.7 and HEK293 cells were purchased from ATCC (Rockville, MD, USA). All other chemicals used in this study were of analytical grade from Sigma Chemical Company. Phospho-specific or total-protein antibodies recognizing p65, p50, inhibitor of κBα (IκBα), Src, spleen tyrosine kinase (Syk), TAK1, c-Jun, c-Fos, ERK, JNK, c-Raf, IRAK1, MEK1/2, MKK4, Myc, HA, lamin A/C, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA).
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