Anti p nfκb p65

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-p-NFκB p65 is a laboratory reagent used to detect and quantify the phosphorylated form of the NF-κB p65 subunit. This antibody can be utilized in various applications, such as Western blotting, ELISA, and immunohistochemistry, to study the activation of the NF-κB signaling pathway.

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7 protocols using anti p nfκb p65

Nuclear and Cytoplasmic Protein Extraction

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Total protein was extracted from PC12 cells using cytoplasmic and nuclear protein extraction kits (CWBIO; Beijing, China). After harvesting the PC12 cells, the cell pellet was resuspended in cytoplasmic protein extraction reagent A and PMSF, and incubated for 10 min on ice. Cytoplasmic protein extraction reagent B and PMSF was added to the cells and mixed, then the tube was centrifuged at 12 000 g for 5 min at 4°C to collect the supernatant comprising the cytoplasmic extract, which was then transferred to a clean pre-chilled tube. The insoluble pellet fraction was resuspended in nuclear protein extraction reagent, and incubated on ice for 30 min. The mixture was then centrifuged at 12 000 g for 10 min at 4°C to collect the supernatant comprising nuclear extract. Cytoplasmic and nuclear protein extracts were separated on SDS-PAGE gels for Western blotting as described above, using anti-p-NFκB p65 (Santa Cruz) to detect phosphorylated p65. Mouse anti-Lamin B (1: 500 dilution, ab16048, Abcam) was used as a nuclear endogenous control and GAPDH was used as the cytoplasmic endogenous control.

Zhang Z., Sun Y, & Chen X. (2020). NLRC5 alleviated OGD/R-induced PC12-cell injury by inhibiting activation of the TLR4/MyD88/NF-κB pathway. The Journal of International Medical Research, 48(8), 0300060520940455.

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Macrophage and Neutrophil Quantification

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Macrophages were detected with an anti-F4/80 antibody (1:50; BD Bioscience, San Jose, CA) and neutrophils were detected with anti-Ly6G antibody (1:50; BD Bioscience) in addition to their respective isotype controls. Following addition of HRP-conjugated secondary antibodies, slides were counter stained with H&E. F4/80 and Ly6G was quantified by digital images of every 5th field of view for the entire colon using Adobe Photoshop as previously described (26 (link)). NF-κB expression was detected by staining with a fluorescently labeled anti-p100/p52 antibody (1:100, Santa Cruz Biotech, TX) or anti-pNF-κB p65 (1:100, Santa Cruz Biotech). Nuclei were counterstained with DAPI. Relative ratios of p52/DAPI were determined using Image J.

Mackos A.R., Allen J.M., Kim E., Ladaika C.A., Gharaibeh R.Z., Moore C., Parry N.M., Boyaka P.N, & Bailey M.T. (2019). Mice Deficient in Epithelial or Myeloid Cell Iκκβ Have Distinct Colonic Microbiomes and Increased Resistance to Citrobacter rodentium Infection. Frontiers in Immunology, 10, 2062.

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Protein Expression Analysis of Mouse Hippocampus

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Homogenize the fresh mouse hippocampal and put it into the lysis buffer. Extract the total protein from the brain tissue, after quantitative determination of protein, SDS-PAGE electrophoresis, membrane transferred, blocked, added primary antibody (including anti-iκB-α (1:500, cell signaling technology, USA), anti-NF-κB p65 (1:1000, cell signaling technology, UK), anti-p-NF-κB p65 (1:1000, Santa Cruz Biotechnology, USA), anti-IL-1β (1:1000, Santa Cruz Biotechnology, USA), anti-TLR4 (1:1000, Santa Cruz Biotechnology, USA) and anti-p-iκB-α (1:200, Santa Cruz Biotechnology, USA)) and then used the appropriate secondary antibody to detect the protein.18 (link) After imaging, the protein of TLR4 and IL-1β expression was expressed by the ratio of the gray value of the target protein to the gray value of GAPDH. The expression of other proteins was expressed by the ratio of the gray value of phosphorylated protein to the gray value of total protein. The Western blot results were analyzed by image lab and Image J, and the experimental data were processed by GraphPad prism 8.

Zhang P., Wang T., Zhu X., Feng L., Wang J., Li Y., Zhang X., Cui T, & Li M. (2023). Jiedu Yizhi Formula Improves Cognitive Function by Regulating the Gut Dysbiosis and TLR4/NF-κB Signaling Pathway. Neuropsychiatric Disease and Treatment, 19, 49-62.

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Molecular Profiling of Intestinal Tumors

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The mucosal lining of Apc^LoxP/+-Cdx2 mice (n=7 per group) and Apc^Min/+ mice (n=3 per group) was stripped for subsequent analysis. The tissue was homogenized in radioimmune precipitation assay buffer with complete Mini protease and PhosSTOP phosphatase inhibitor (Roche Diagnostics, Mannheim, Germany). The tissue lysates were separated on polyacrylamide gels (BioRad) for Western blot analysis. The following primary antibodies were used: anti-COX2, anti-Bcl-2, anti-β-catenin, anti-EGFR, anti-p-EGFR, and anti-Cyclin D1 (Cell Signaling, Beverly, MA), anti-p-NFκB p65, and anti-RXRα (Santa Cruz, Dallas, TX). Goat anti-mouse (1:6000) or goat anti-rabbit (1:6000) secondary antibodies were used (Abcam, Cambridge, MA). The membranes were detected by the Enhanced Chemiluminescence Detection kit (GE Healthcare, Chicago, IL)

Bowen C.M., Walter L., Borras E., Wu W., Ozcan Z., Chang K., Bommi P.V., Taggart M.W., Thirumurthi S., Lynch P.M., Reyes-Uribe L., Scheet P.A., Sinha K.M, & Vilar E. (2021). Combination of Sulindac and Bexarotene for Prevention of Intestinal Carcinogenesis in Familial Adenomatous Polyposis. Cancer prevention research (Philadelphia, Pa.), 14(9), 851-862.

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Immunoblot Analysis of Key Signaling Proteins

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After homogenization of raw 264.7 cells, proteins were isolated using a protein lysis buffer (PRO-PREP™, Intron Biotechnology, Seongnam, Republic of Korea) and subjected to immunoblot analysis according to the manufacturer’s instructions. Whole proteins were separated on SDS-PAGE gels and immunoblotted using primary antibodies. The primary antibodies used are as follows: (1) anti-CREB and anti-p-CREB (1:1000, Cell Signaling Technology, MA); (2) anti-NF-κB p65 and anti-p-NF-κB p65 (1:1000, Santa Cruz Biotechnology, TX), and (3) anti-IL-1ra (1:500, Santa Cruz Biotechnology, TX). GAPDH (1:5000, Santa Cruz Biotechnology, TX) was used as an internal control. Quantification of the bands was performed using the NIH ImageJ program. Independent experiments were performed in triplicate on different days.

Oh S.H., Choi C., Noh J.E., Lee N., Jeong Y.W., Jeon I., Shin J.M., Kim J.H., Kim H.J., Lee J.M., Kim H.S., Kim O.J, & Song J. (2018). Interleukin-1 receptor antagonist-mediated neuroprotection by umbilical cord-derived mesenchymal stromal cells following transplantation into a rodent stroke model. Experimental & Molecular Medicine, 50(4), 22.

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Western Blot Analysis of NF-κB p65 Activation

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Total protein was quantitated using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Equal quantities of total protein (20 μg) were separated on 10% Bis-Tris gels in MOPS SDS Running Buffer (Invitrogen Life Technologies, Carlsbad, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon; Millipore Corporation, Bedford, MA, USA). The PVDF membranes were blocked with 5% skim milk in Tris-buffered saline [TBS; 50 mM Tris-Cl (pH 7.5), 150 mM NaCl] for 60 min at room temperature and then incubated overnight with anti-pNF-κB p65 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or anti-β-actin antibodies (1:500; Santa Cruz Biotechnology, Inc.) in TBS-Tween-20 (TBST; 0.1% Tween-20). The membranes were washed three times with 1X TBST for 5 min, and then incubated for 1 h with secondary antibodies conjugated to horseradish peroxidase (HRP) at room temperature. The membrane was exposed to high performance autoradiography film (Fuji XR film; Fujifilm Corporation, Tokyo, Japan) and visualized using an enhanced chemiluminescence system (Santa Cruz Biotechnology, Inc.).
Integrated density values of the band intensities from the films were analyzed by ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA).

WANG H., XU L., ZHAO J., WANG D., GUO R., WANG J., GONG W., LIU T., ZHANG Y, & DONG L. (2014). Regulatory mechanism of pyrrolidine dithiocarbamate is mediated by nuclear factor-κB and inhibits neutrophil accumulation in ARDS mice. Experimental and Therapeutic Medicine, 8(2), 614-622.

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Anti-Inflammatory Signaling Mechanisms

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Dulbecco's Modified Eagle's Medium was purchased from Welgene (Gyeongsan, Korea) and fetal bovine serum (FBS) from Gibco BRL (Gaithersburg, MD, USA). ELISA kits for IL-1β, TNF-α, and IL-6 were obtained from Ab Frontier (Seoul, Korea) and PGE₂ was purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies, that is, anti-HO-1, anti-COX-2, anti-iNOS, anti-p-IκB-α, anti-p-NF-κB (p65), and anti-Nrf2 and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dimethyl sulfoxide (DMSO) was purchased from Junsei Chemical Co. (Tokyo, Japan), and LPS (E. coli 055:B5), geniposide, puerarin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Griess reagent, 4,6-diamidino-2-phenylindole (DAPI), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Lee S.C., Kwon Y.W., Park J.Y., Park S.Y., Lee J.H, & Park S.D. (2017). Antioxidant and Anti-Inflammatory Effects of Herbal Formula SC-E3 in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 1725246.

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