Anti nf κb p65 rabbit mab

Manufactured by Cell Signaling Technology

Anti-NF-κB p65 rabbit mAb is a laboratory reagent used to detect and study the NF-κB p65 subunit. It is a highly specific monoclonal antibody produced in rabbits.

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Lab products found in correlation

Anti rabbit phospho nf κb p65 rabbit antibody, by Cell Signaling Technology (2 mentions) Lps from escherichia coli 055 b5, by Merck Group (1 mentions) Bay 11 7082, by Beyotime (1 mentions) Anti bcl 2 rabbit mab, by Cell Signaling Technology (1 mentions) Anti parp rabbit mab, by Cell Signaling Technology (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Anti cd31 antibody, by Santa Cruz Biotechnology (1 mentions) Tcs sp5, by Leica (1 mentions) Cl xposure film, by Thermo Fisher Scientific (1 mentions) Cell fractionation kit, by Cell Signaling Technology (1 mentions) Goat anti rabbit igg hrp linked ab, by Cell Signaling Technology (1 mentions) Supersignal west femto maximum sensitivity substrate solution, by Thermo Fisher Scientific (1 mentions) Kwikquant ultra digital ecl substrate solution, by Kindle Biosciences (1 mentions) Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

NF-κB p65 (724 citations) NF-κB (393 citations) Anti-NF-κB p65 (293 citations) Anti-p65 (276 citations) Anti-NF-κB (140 citations) Rabbit anti-NF-κB p65 (60 citations) Rabbit anti-p65 (44 citations) Rabbit anti-NF-κB (16 citations) NF-κB p65 rabbit mAb (11 citations) Anti-NF-κB p65 rabbit mAb (D14E12, (3 citations)

4 protocols using anti nf κb p65 rabbit mab

Pulmonary Endothelial Cell Inflammatory Regulation

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For cell culture and reagents, primary mouse pulmonary microvascular endothelial cells were obtained from Angio-Proteomie (Boston, MA, US) and maintained in Angio-Proteomie Endothelial Cell Medium in a humidified 37°C, 5% CO₂ incubator. The medium was changed at 48-hour intervals. Genistein-3′-sodium sulfonate (GSS) was obtained from Shanghai Tianxi Chemical Co., Ltd. (Shanghai, China), and BAY11-7082 was purchased from Beyotime of China. The LPS (from Escherichia coli 055:B5) was obtained from Sigma-Aldrich (St. Louis, MO). The anti-GEF-H1 rabbit monoclonal antibody (mAb), anti-Myd88 rabbit mAb, anti-NF-κB p65 rabbit mAb, and anti-rabbit phospho-NF-κB-p65 rabbit antibody were obtained from Cell Signaling Technologies (Danvers, MA). Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody and the ProLong Gold Antifade Mountant with DAPI were obtained from Invitrogen Life Science.

Yi L., Zhou Z., Zheng Y., Chang M., Huang X., Guo F., Zhao Q, & Huan J. (2019). Suppressive Effects of GSS on Lipopolysaccharide-Induced Endothelial Cell Injury and ALI via TNF-α and IL-6. Mediators of Inflammation, 2019, 4251394.

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Primary Mouse Pulmonary Microvascular Endothelial Cell Isolation

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Primary mouse pulmonary micro‐vascular endothelial cells (MPMECs) were obtained Angio‐Proteomie and maintained in Angio‐Proteomie Endothelial Cell Medium in a humidified 37°C, 5% CO₂ incubator. GSS was obtained from Shanghai Tian Xi Chemical Co., Ltd., and BAY11‐7082 was purchased from Beyotime of China. The LPS was obtained from Sigma‐Aldrich. Anti‐GEF‐H1 rabbit monoclonal antibody (mAb), anti‐Myd88 rabbit mAb, anti‐NF‐κB p65 rabbit mAb, anti‐rabbit phospho‐NF‐κB‐p65 rabbit antibody, anti‐BCL‐2 rabbit mAb and anti‐PARP rabbit mAb were obtained from Cell Signaling Technology. Anti‐CD31 antibody and anti‐cleaved‐PARP antibody were purchased from Santa Cruz Biotechnology. The Alexa Fluor 555‐conjugated goat antimouse secondary antibody and Alexa Fluor 594‐conjugated goat anti‐rabbit secondary antibody and the ProLong Gold Antifade Mountant with DAPI were obtained from Invitrogen Life Sciences.

Yi L., Chang M., Zhao Q., Zhou Z., Huang X., Guo F, & Huan J. (2019). Genistein‐3′‐sodium sulphonate protects against lipopolysaccharide‐induced lung vascular endothelial cell apoptosis and acute lung injury via BCL‐2 signalling. Journal of Cellular and Molecular Medicine, 24(1), 1022-1035.

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Visualizing NF-κB Activation in LPS-Treated RAW264.7 Cells

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RAW264.7 cells were plated at a density of 3 × 10⁴ cells/well in 24-well plates with coverglass slides and then treated with 20 ng/ml LPS P. aeruginosa ATCC 27316 serotype 10 (Sigma-Aldrich, L9143) in the presence of 20 μm SET-M33 for 6 h at 37 °C, fixed by incubation with a PBS-4% paraformaldehyde solution for 15 min, saturated for 60 min at room temperature with PBS, 5% normal bovine serum, and 0.3% TritonX-100, and incubated with anti NF-κBp65 rabbit mAb (Cell Signaling Technology) diluted 1:50 in PBS-1% BSA, 0.3% Triton X-100 overnight at 4 °C and then with anti-rabbit-FITC (Sigma-Aldrich) for 1 h at room temperature. Membranes were stained with Lectin Atto-647. NF-κB expression was analyzed by confocal laser microscope (Leica TCS SP5) with 488/520- to 540-nm excitation and 633/660- to 690-nm emission filters for FITC and Atto-647, respectively. All images were processed using ImageJ software (National Institutes of Health).

Brunetti J., Roscia G., Lampronti I., Gambari R., Quercini L., Falciani C., Bracci L, & Pini A. (2016). Immunomodulatory and Anti-inflammatory Activity in Vitro and in Vivo of a Novel Antimicrobial Candidate. The Journal of Biological Chemistry, 291(49), 25742-25748.

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Western Blot Analysis of Subcellular Fractions

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Subcellular fractions were isolated using Cell Fractionation kit (#9038S, Cell Signaling Technology) followed the manufacturer’s manual. 10 μg of nuclear fractions were loaded to each well of Bolt 4–12% Bis-Tris Plus Gels (# NW04125BOX, Thermo Fisher Scientifics) for SDS-PAGE and transferred onto 0.45 μm PVDF membranes (#IPVH15150, Millipore). As primary antibodies anti-Histone H3 rabbit polyclonal Ab (#07–690, Sigma-Aldrich, 1:2000), anti-Lamin B1 Rabbit mAb (HRP Conjugate) (#15068S, Cell Signaling Technology, 1:5000), anti-NF-κB p65 rabbit mAb (#8242S, Cell Signaling Technology, 1:1000), anti-Bsg rabbit mAb (#MA5–32534, Thermo Fisher Scientific, 1:1000) and anti-Hk2 rabbit mAb (#2867, Cell signaling Technology, 1:1000) were used, followed by goat anti-rabbit IgG HRP linked Ab (#7074S, Cell Signaling Technology, 1:2000). Blots were developed using the SuperSignal West Femto Maximum sensitivity Substrate Solution (#34095, Thermo Fisher Scientific) or KwikQuant Ultra Digital-ECL Substrate Solution (#R1002, Kindle Biosciences). CL-XPosure Film (#34090, Thermo Fisher scientific) or KiwiQuant® imager (#D1001, Kindle Biosciences) were used for signal detection. Data quantification was done using Fiji software (Schindelin et al., 2012 (link)).

Chao C.C., Gutiérrez-Vázquez C., Rothhammer V., Mayo L., Wheeler M.A., Tjon E.C., Zandee S.E., Blain M., De Lima K.A., Takenaka M.C., Avila-Pacheco J., Hewson P., Liu L., Sanmarco L.M., Borucki D.M., Lipof G.Z., Trauger S.A., Clish C.B., Antel J.P., Prat A, & Quintana F.J. (2019). Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS. Cell, 179(7), 1483-1498.e22.

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