Western blot was performed as described previously (5 (link),13 (link),33 (link)). Briefly, the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto Hybond ECL membranes (Amersham Pharmacia, Piscataway, NJ). The ECL membranes were incubated with the appropriate primary antibody anti-IRAK1 (sc-7883, Santa Cruz Biotechnology), anti-TRAF6 (sc-7221, Santa Cruz Biotechnology), and anti-IκBα, respectively, followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc.) and analysis by the ECL system (Amersham Pharmacia, Piscataway). The signals were quantified using the G:Box gel imaging system by Syngene (Syngene, USA, Fredrick, MD).
Anti irak1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-IRAK1 is a laboratory reagent used for the detection and quantification of IRAK1 (Interleukin-1 Receptor-Associated Kinase 1) in various biological samples. It is a primary antibody that specifically binds to IRAK1, a key signaling molecule involved in the inflammatory response. The core function of Anti-IRAK1 is to enable researchers to analyze the expression and/or activity of IRAK1 in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti traf6, by Santa Cruz Biotechnology (4 mentions)
Anti iκbα, by Cell Signaling Technology (4 mentions)
Ecl system, by Cytiva (2 mentions)
Anti ikkβ, by Cell Signaling Technology (2 mentions)
Anti phospho ikkβ, by Cell Signaling Technology (2 mentions)
Anti tak1, by Santa Cruz Biotechnology (2 mentions)
Anti p p65, by Cell Signaling Technology (2 mentions)
Anti p38, by Cell Signaling Technology (2 mentions)
Anti p ikkα β, by Cell Signaling Technology (2 mentions)
Anti p p38, by Cell Signaling Technology (2 mentions)
Anti p iκbα, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hybond ecl membrane, by Cytiva (1 mentions)
Peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
G box gel imaging system, by Syngene (1 mentions)
Bh3 mjl, by Olympus (1 mentions)
Goat anti rabbit igg cfl 555, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Z vrpr fmk, by Enzo Life Sciences (1 mentions)
15 protocols using anti irak1
1
Western Blot Analysis of Inflammatory Signaling
2
Immunofluorescence Staining of IRAK-1
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Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) and 0.1% Triton X-100 for 1 h and incubated with anti-IRAK-1 (Santa Cruz Biotechnology, Inc.) at 4°C overnight. Cells were washed with PBS and incubated with goat anti-rabbit IgG-CFL 555 (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Cells were washed with PBS and stained with DAPI for 15 min at room temperature. Then, cells were washed with PBS and observed under a light microscope (magnification, ×100, BH3-MJL; Olympus Corporation).
3
Antibodies and Inhibitors for Signaling Pathways
4
Immunoblotting Analysis of Mouse Lung Proteins
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Mouse lung tissue homogenates were separated by electrophoresis on 10 to 15% SDS-PAGE, transferred on PVDF membranes (Merck Millipore, Burlington, MA, USA), blocked in 5% of non-fat dry milk, and incubated with primary antibodies including anti-CitH3 (1:1000, citrulline R2 + R8 + R17, Abcam, Cambridge, UK), anti-TRAF6 (1:1000, Santa Cruz), anti-IRAK-1 (1:1000, Santa Cruz), anti-PAD4 (1:1000, Abcam), anti-HMGB1 (1:1000, Abcam) or anti-β-actin antibodies (1:5000, Sigma-Aldrich) at 4 °C for 18 h. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch, West Grove, PA, USA) at RT for 2 h. Signal expression of protein-antibody complexes was detected by ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and visualized with Biospectrum imaging system (UVP, Upland, CA, USA). Relative protein expression levels were measured by Image J (National Institute of Health, Bethesda, MD, USA).
5
IRAK1 Signaling Pathway Immunoprecipitation
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Cells were lysed in an endogenous lysis buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 30 mM NaF, and 2 mM sodium pyrophosphate) supplemented with complete protease inhibitor cocktail (Roche), phosphatase inhibitor tablet (Roche), 1 mM DTT, 1 mM Na3VaO4, and 1 mM PMSF. Samples were cleared by centrifugation at 14,000 rpm, and protein concentration was measured using the BCA Protein Assay kit (Thermo Fisher Scientific). For immunoblotting, total proteins were separated on 4–12% SDS–polyacrylamide gels and transferred to nitrocellulose membranes. For immunoprecipitation experiments, samples were first precleared with Protein G Dynabeads (Invitrogen) for 60 min. Cleared lysates were incubated overnight with an anti-IRAK1 antibody (Santa Cruz Biotechnology, Inc.), followed by Protein G Dynabeads for an additional 2 h at 4°C. Immunoprecipitates were washed five times with 0.5 M NaCl lysis buffer, separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting. Antibodies used were as follows: anti-IKKβ, anti–phospho-IKKβ, anti–phospho-IRAK4 (pThr-345/Ser-346), and anti-IRAK4 (total) from Cell Signaling Technologies; and anti–β-actin and anti-IRAK1 from Santa Cruz Biotechnology, Inc. Secondary HRP-conjugated antibodies were obtained from GE Healthcare.
6
Immunoprecipitation and Immunoblotting Protocols
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Peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Millipore (Billerica, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), fetal calf serum, and lipofectamine and PLUS reagents were purchased from Life Technologies (Grand Island, NY, USA). Anti-Myc, anti-GAPDH, anti-TAK1, anti-TRAF6 and anti-IRAK1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-RCAN1 antibodies were purchased from ECM Biosciences (Versailles, KY, USA) and Santa Cruz Biotechnology. Anti-LRRK2, anti-phospho-LRRK2 (Ser935), and anti-phospho-TAK1 (Thr187) antibodies were purchased from Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively. Polyclonal and monoclonal anti-HA antibodies were purchased from Abnova (Tebu, France) and Covance (Powhatan, VA, USA), respectively. Protein A-Sepharose beads and glutathione sepharose 4B were purchased from GE Healthcare Life Science (Piscataway, NJ, USA). Enhanced chemiluminescence (ECL) reagent and [γ-32P] ATP were purchased from Perkin Elmer Life Sciences (Downers Grove, IL, USA). MG132 was purchased from A.G. Scientific (San Diego, CA, USA). Human recombinant IL-1β was purchased from Sigma (St. Louis, MO, USA).
7
Isolation and Culture of Splenic Macrophages
8
Regulation of IRF7 Signaling Pathway
9
Immunohistochemical Analysis of IRAK1, Vimentin, and IL6
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Harvested tissues were fixed in 10% formalin solution, (HT501128, Sigma-Alrich). Tissues were dehydrated and embedded in paraffin. Paraffin-embedded tissue sections (5 μm thick) were cut, deparaffinized and rehydrated, and antigens were retrieved using pH 6 sodium citrate. The sections were then incubated in 0.06% hydrogen peroxide at room temperature, to block endogenous peroxidase. The slides were incubated overnight with anti-IRAK1 (catalogue number: sc-7883, 1:100 dilution), anti-vimentin (catalogue number: sc-6260, 1:500 dilution) from Santa Cruz Biotechnology and anti-IL6 from Abcam (Cambridge, MA; catalogue number: ab1543670, 1:1000 dilution) overnight, followed by 60 min incubation with anti-mouse IgG/rabbit IgG (catalogue number: PK-6200, 1:2000 dilution) and 60 min of Avidin DH and Biotinylated Horseradish Peroxidase H. ImmPACT DAB Peroxidase Substrate (catalogue number: SK-4105) was used as the chromogen. Vector Hematoxylin QS (H-3404) was used as counterstain.
10
TLR2 Signaling Pathway Activation
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