Nf κb

Manufactured by Merck Group

Sourced in United States

NF-κB is a laboratory equipment product that functions as a transcription factor. It plays a crucial role in regulating gene expression, particularly in response to various cellular stimuli, such as inflammation, stress, and immune responses.

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NF-κB inhibitor Bay 11-7082 (15 citations) NF-κB p65 (15 citations) NF-κB inhibitor (10 citations) NF-κB SN50 inhibitor (5 citations) Anti-NF-κB p65 (3 citations) NF-κB activation inhibitor (2 citations) Anti-p65-NF-κB (C-20) (2 citations) Rabbit anti-p65 NF-κB antibody (2 citations) 5′-biotin labeled NF-κB oligo (2 citations) NF-κB SN50 (2 citations)

21 protocols using nf κb

TNBS-induced Inflammation Model

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TNBS was purchased from Sigma (St. Louis, MO, USA) and dissolved in ethanol. Mouse monoclonal antibody against nuclear factor-κB (NF-κB) and rabbit polyclonal antibody against interleukin-1β (IL-1β) were purchased from Chemicon (Temecula, CA, USA) and Santa Cruz (Santa Cruz, CA, USA), respectively.

Ho T.Y., Lo H.Y., Chao D.C., Li C.C., Liu J.J., Lin C, & Hsiang C.Y. (2014). Electroacupuncture Improves Trinitrobenzene Sulfonic Acid-Induced Colitis, Evaluated by Transcriptomic Study. Evidence-based Complementary and Alternative Medicine : eCAM, 2014, 942196.

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Immunohistochemical Analysis of NF-κB and β-Catenin

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Paraffin-embedded infected ileal sections were deparaffinized in xylene and dehydrated through graded concentrations of isopropyl alcohol. After blocking the endogenous peroxidase activity with 3% H₂O₂, the sections were heated in 10 mM sodium citrate buffer at pH 6.0 in a microwave oven for 20 min. The slides were allowed to cool at room temperature and non-specific binding was blocked with 3% BSA for 1 hr at room temperature. The sections were incubated with primary antibodies NF-κB (dilution 1:250; Chemicon) and β-catenin (dilution 1:200; Santa Cruz, USA) overnight at 4°C in a humidified chamber. Bound antibody was detected by a horseradish peroxidase-conjugated secondary antibody. The peroxidase reaction was developed in PBS with hydrogen peroxide as substrate and diaminobenzidine (DAB) as a chromogen. Sections were counterstained with Mayer’s hematoxylin, rehydrated and mounted with DPX and then visualized under Axioskope 2d microscope, Carl Zeiss, Germany.

Gopal A., Chidambaram I.S., Devaraj N, & Devaraj H. (2017). Shigella dysenteriae infection activates proinflammatory response through β-catenin/NF-κB signaling pathway. PLoS ONE, 12(4), e0174943.

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Antibody Acquisition for Metabolic Assays

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Naringin (purity >90%), DHE (CAS Number: 104821-25-2) and the primary antibody against DGAT2 (Product number: SAB2106887, Lot number: QC29804) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Primary antibodies against ACC (CAS Number: 05-1098), Nrf2 (CAS Number: MABE1799), HO-1 (CAS Number: AB1284), NF-κB (CAS Number: ABE347), TNF-α (CAS Number: AB 1837P) and Lamin B1 (CAS Number: MABS492) were purchased from Merck KGaA (Darmstadt, Germany). The primary antibody against ChREBP (Product number: OAAN03715) was purchased from Aviva System Biology (San Diego, CA, United States). The primary antibodies against SREBP-1c (CAS Number: PA1-46142), FAS (CAS Number: MA5-14887) and β-actin (CAS Number: MA5-15739) were purchased from Thermo Fisher Scientific (Waltham, MA, United States). The primary antibodies against GPAT-1 (CAS Number: ab69990) was purchased from Abcam (Waltham, MA, United States).

Pengnet S., Sumarithum P., Phongnu N., Prommaouan S., Kantip N., Phoungpetchara I, & Malakul W. (2022). Naringin attenuates fructose-induced NAFLD progression in rats through reducing endogenous triglyceride synthesis and activating the Nrf2/HO-1 pathway. Frontiers in Pharmacology, 13, 1049818.

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Neuroinflammation Markers Profiling Protocol

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6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381)

β-Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441)

Suramin (Sigma, St. Louis, MO, USA; catalog: S2671)

iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600)

COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126)

NF-κB (Merck Millipore, Massachusetts, USA; catalog: MAB3026)

Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029)

Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP)

PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP)

Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588)

Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP)

Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101)

Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A)

Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Feng C.W., Chen N.F., Chan T.F, & Chen W.F. (2020). Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo. Parkinson's Disease, 2020, 8814236.

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Immunoblot and Cytometric Analysis of Immune Markers

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Antibodies used in this study for immunoblot include: anti-phospho-ERK1/2 (#4370, Cell Signaling Technologies), anti-ERK1/2 (sc-514302, Santa Cruz), anti-Annexin A1 (#71-3400, Invitrogen), anti-β-actin (A5316, Sigma-Aldrich), and HRP-labeled secondary goat anti-mIgG (#31430, Invitrogen) and anti-rIgG (#31460, Invitrogen). Antibodies used in this study for flow cytometric staining and/or immunohistochemistry staining were: fluorescein-labeled anti-B220 (#103207, clone RA3-6B2, Biolegend), anti-CD19 (#115508, clone 6D5, Biolegend), anti-CD3e (#553061, clone 145-2C11, BD Biosciences), anti-CD11c (#117313, clone N418, Biolegend), anti-CD103 (#121413, clone 2E7, Biolegend), anti-IgD (#17-5993-82, clone 11–26, eBioscience), anti-CD3 (#100240, clone 17A2, Biolegend), anti-B220 (#103228, clone RA3-6B2, Biolegend) antibodies, and Hoechst 33,342 nucleic acid staining reagent (#H3570, Invitrogen) as well as HRP-labeled anti-NFκB (#MAB3026, Merck) antibody.
Genomic DNA extraction kit (#KN-T110005, KANEKA Co., Japan) and Taq DNA polymerase (#M0273, NEB Biolabs) were used for mice genotyping. All other biochemicals were purchased from Sigma-Aldrich.

Yap J., Yuan J., Ng W.H., Chen G.B., Sim Y.R., Goh K.C., Teo J., Lim T.Y., Goay S.M., Teo J.H., Lao Z., Lam P., Sabapathy K, & Hu J. (2023). BRAF(V600E) mutation together with loss of Trp53 or pTEN drives the origination of hairy cell leukemia from B-lymphocytes. Molecular Cancer, 22, 125.

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Immunofluorescence Analysis of Inflammatory Markers

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Tissue sections were deparaffinized, rehydrated, through a series of graded ethanol (100, 95, 70, 50, and 30%) and water, followed by an antigen retrieval step by heating the sections in microwave apparatus using a glass container containing 10 mM sodium citrate buffer, pH 6.0 for 10 min. Sections were then cooled at room temperature, incubated in blocking solution (3% bovine serum albumin (BSA) with 0.2% Tween 20 in PBS for 1 h, followed by incubation overnight at 4 °C with primary antibodies: MUC1 (1:400 dilution, Abcam), MUC2 (1:200 dilution, Abcam), iNOS (1:200 dilution, BD Bioscience), p47^PHOX (1:500 dilution, Santa Crux), TRL4 (1:200 dilution, Santa Crux) and NF-κB (1:100 dilution, EMD Millipore) diluted in 3% blocking solution. Next day, sections were washed and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:400 dilution, Abcam) and/or Alexa Fluor 647 (1:400 dilution, Abcam) for 1 h at room temperature in the dark chamber. The sections were washed in PBS and rinsed in distilled water and mounted using Fluroshield mounting medium with 4’, 6-diamidino-2-phenylindole (DAPI) (Abcam). Images were captured with a microscope (OLYMPUS BX51) under fluorescence setup with appropriate filters.

Periasamy S., Lin C.H., Nagarajan B., Sankaranarayanan N.V., Desai U.R, & Liu M.Y. (2018). Mucoadhesive role of tamarind xyloglucan on inflammation attenuates ulcerative colitis. Journal of functional foods, 47, 1-10.

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Western Blot Analysis of Lung Proteins

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Protein samples were prepared from the lung tissues using 1× cell lysis buffer (Cell Signalling Technology, USA). Equivalent amounts of proteins (10–20 µg) were used to assess protein expressions as described previously [11 (link),12 (link)]. Briefly, protein was denatured by boiling, separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE, 12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 8% non-fat milk/PBS-T (PBS with 0.5% Tween-20) for 2 h and respectively incubated with the following rabbit-derived primary antibodies: NF-κB, Bax, Bcl-xL, cleaved-Caspase-3 and β-actin (all 1:1000 dilution; Sigma Aldrich, St. Louis, MO, USA). After washing, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000 dilution; Cell Signalling Technology, USA). Proteins of interest were visualised using the enhanced chemiluminescence kit (EMD Millipore, USA), and the band intensities were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Zhang L., Tai Q., Xu G, & Gao W. (2021). Lipoxin A4 attenuates the lung ischaemia reperfusion injury in rats after lung transplantation. Annals of Medicine, 53(1), 1143-1152.

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Western Blot Analysis of Protein Signaling

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Protein (50 μg) was separated by 10% and 12% SDS-polyacrylamide gel electrophoresis, based on the molecular weight of the protein of interest and transferred onto a nitrocellulose membrane (Millipore, Billerica, USA). The membranes were blocked with 3% bovine serum albumin in PBS containing 0.1% Tween 20 (Sigma), washed and probed with respective mouse/rabbit monoclonal antibodies. Primary antibodies PDI, Akt, p-Akt and CHOP/GADD153 were obtained from Santa Cruz Biotech, p70S6kinase from Cell Signaling Technology (MA, USA), GRP-78, GSK-3β and NFκB from Sigma (St. Louis, MO, USA) while MAFbx-1 and MuRF-1 from abcam. The membranes were then incubated with anti-mouse/rabbit-IgG HRP conjugate (Sigma). The membrane was washed and incubated with chemiluminescent substrate (Sigma) and the bands were developed using Gel Documentation System (UVP Bioimaging software, Upland CA, USA). Quantification was performed by densitometry using ImageJ software. GAPDH from Sigma (St. Louis, MO, USA) was used as an internal (loading) control.

Agrawal A., Rathor R., Kumar R., Suryakumar G, & Ganju L. (2018). Role of altered proteostasis network in chronic hypobaric hypoxia induced skeletal muscle atrophy. PLoS ONE, 13(9), e0204283.

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Protein Expression Analysis in Lung Tissues

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Proteins were extracted from lung tissues, and their concentrations were measured using the Bradford assay. Proteins were separated using a polyacrylamide gel followed by transfer to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with primary antibodies including iNOS, HO-1, NF-κB, Bax, and Bcl-xL (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C, followed by incubation with the appropriate secondary antibodies (Santa Cruz Biotechnology, Oregon, USA). An enhanced chemiluminescence reagent (Santa Cruz Biotechnology Oregon, USA) was then added to visualize the proteins.

Ju Y.N., Tai Q.H., Xu G.X., Zhao X.Q., Sun H.B, & Gao W. (2021). Diannexin Can Ameliorate Acute Respiratory Distress Syndrome in Rats by Promoting Heme Oxygenase-1 Expression. Mediators of Inflammation, 2021, 1946384.

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Western Blot Detection of MAPK and NF-κB Signaling

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Detections of phospho-p44/42 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, and NF-κB p65 were performed using western blot. After PA treatment, cells lysed in buffer containing 1% Triton X-100 (Sigma-Aldrich) and Protease/Phosphotase Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). Protein concentrations assessed with Quick Start Bradford Protein Assay kit following manufacturer's protocol. Lysates loaded with Laemmli Sample Buffer onto Any kD Mini-PROTEAN-TGX pre-cast polyacrylamide gel (Bio-Rad) and separated by electrophoresis. Gels transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) and detected with primary antibodies for MAPK (Cell Signaling) or NF-κB (Sigma-Aldrich) pathways, secondary horse radish peroxidase-conjugated antibody (Abcam, Cambridge, England), and chemiluminescent substrate (Thermo Fisher Scientific) before visualization with ChemiDoc MP System (Bio-Rad).

Park A.J., Agak G.W., Qin M., Hisaw L.D., Pirouz A., Kao S., Marinelli L.J., Garbán H.J., Thiboutot D., Liu P.T, & Kim J. (2017). G2A Attenuates Propionibacterium acnes Induction of Inflammatory Cytokines in Human Monocytes. Annals of Dermatology, 29(6), 688-698.

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