Nf κb p65 d14e12 xp rabbit mab

Manufactured by Cell Signaling Technology

Sourced in United States

The NF-κB p65 (D14E12) XP® Rabbit mAb is a primary antibody that recognizes the p65 subunit of the NF-κB transcription factor. It is designed for use in applications such as western blotting, immunoprecipitation, and immunohistochemistry to detect the endogenous levels of p65 protein.

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Lab products found in correlation

Imagequant las 4000 mini, by GE Healthcare (1 mentions) Phospho nf kb p65ser536 93h1 rabbit mab, by Cell Signaling Technology (1 mentions) Ecl chemiluminescent detection kit, by Merck Group (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Trypsin edta solution, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Phenazine methosulfate, by Merck Group (1 mentions) Celltiter96 aqueous non radioactive cell proliferation assay mts kit, by Promega (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Ab32518, by Cell Signaling Technology (1 mentions) Ab137040, by Cell Signaling Technology (1 mentions) Ab133741, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Goat anti rabbit igg h l hrp, by Abcam (1 mentions) Neuronal nuclei (neun), by Synaptic Systems (1 mentions) Ab53554, by Abcam (1 mentions) Ab6142, by Abcam (1 mentions) Ab182451, by Abcam (1 mentions) Ab13970, by Synaptic Systems (1 mentions)

Spelling variants of this product

NF-κB p65 (724 citations) NF-κB (393 citations) XP® Rabbit mAb (21 citations) NF-κB p65 rabbit mAb (11 citations) NF-κB p65 (D14E12) (10 citations) D14E12)XP® Rabbit mAb (5 citations) NF-κB p65 [D14E12] rabbit mAb (3 citations) NF-κB p65 (D14E12) XP® (2 citations) Rabbit mABs (2 citations)

7 protocols using nf κb p65 d14e12 xp rabbit mab

NF-κB Signaling Regulation in M0 THP-1 Macrophages

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The M0 THP-1 macrophages were treated with 5% QPD or 5% XBD for 24 h. Then the cells were stimulated with or without 1 µg/ml LPS and 20 ng/ml IFN-γ. The total intracellular protein was collected at 1 h and 2 h post-stimulation. IκBα, phospho-IκBα, NF-κB p65, and phospho-NF-κB p65 protein levels were assessed by western blot using IκBα (L35A5) mouse mAb, phospho-IκBα (Ser32/36)(5A5) mouse mAb, NF-κB p65 (D14E12) XP^® rabbit mAb, and phospho-NF-kB p65 (Ser536) (93H1) rabbit mAb (Cell Signaling Technology, United States), respectively. Secondary antibodies were HRP-labeled goat anti-mouse or anti-rabbit IgG (Proteintech, China). The protein bands were visualized using an ECL chemiluminescent detection kit (Millipore, United States) in an ImageQuant LAS 4000mini (GE, United States).

Li Y., Li B., Wang P, & Wang Q. (2021). Traditional Chinese Medicine, Qingfei Paidu Decoction and Xuanfei Baidu Decoction, Inhibited Cytokine Production via NF-κB Signaling Pathway in Macrophages: Implications for Coronavirus Disease 2019 (COVID-19) Therapy. Frontiers in Pharmacology, 12, 722126.

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NF-κB Pathway Activation Assay

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A PierceTM BCA Protein Assay Kit, RPMI1640 medium, DPBS/MODIFIED, Trypsin-EDTA Solution, fetal bovine serum, and dual antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). A CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS) kit was obtained from Promega (Madison, WI, USA) and phenazine methosulfate was purchased from Sigma-Aldrich (St. Louis, MO, USA). High Efficiency RIPA tissue/cell rapid lysis solution was acquired from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All antibodies were purchased from Abcam (Cambridge, MA, USA) or Cell Signaling Technology (Beverly, MA, USA) as follows: Nuclear Factor KappaB (NF-κB) p65 (D14E12) XP Rabbit mAb (#8242, dilution 1/1000), p-NF-κB p65 (Ser536) (93H1) rabbit mAb (#3033, dilution 1/1000), anti-IκBα antibody (ab32518, dilution 1/1000), anti-SDHA antibody (ab137040, dilution 1/1000), and anti-Lamin B antibody (ab133741, dilution 1/1000).

Chen B., Chen H., Qu H., Qiao K., Xu M., Wu J., Su Y., Shi Y., Liu Z, & Wang Q. (2022). Photoprotective effects of Sargassum thunbergii on ultraviolet B-induced mouse L929 fibroblasts and zebrafish. BMC Complementary Medicine and Therapies, 22, 144.

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Protein Expression Analysis in Mouse Liver

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Western blotting was performed as previously described.18 Proteins were extracted from liver tissues using RIPA buffer containing PMSF, and protein concentrations were quantified with the bicinchoninic acid (BCA) assay method using a BCA protein assay kit (Thermo Scientific). Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with Tris-buffered saline containing 5% non‑fat milk at 37°C for 2 h. The membranes were then incubated with the following primary antibodies at 37°C for 1 h: α-SMA mAb (14395-1-AP; Proteintech, USA), IL-1β (D6D6T) rabbit mAb (mouse-specific) (#31202; Cell Signaling Technology), TNF-α (D2D4) XP rabbit mAb (mouse-specific) (#11948; Cell Signaling Technology), NF-κB p65 (D14E12) XP rabbit mAb (#8242; Cell Signaling Technology), β-actin (#4970; Cell Signaling Technology), Histone-H3 Antibody125 Publications (17168-1-AP; Proteintech, USA), or anti-IKB alpha [E130] (ab32518; Abcam). Goat anti-rabbit IgG H&L (HRP) (ab205718; Abcam) was used as the secondary antibody. After washing with PBS, the membranes were incubated with the secondary antibodies at 37°C for 1 h. The membranes were developed with an enhanced chemiluminescence (ECL) system (ProteinSimple, Santa Clara, CA, USA).

Luo Y., Yang P., Li Z., Luo Y., Shen J., Li R., Zheng H., Liang Y, & Xia N. (2019). Liraglutide Improves Non-Alcoholic Fatty Liver Disease In Diabetic Mice By Modulating Inflammatory Signaling Pathways. Drug Design, Development and Therapy, 13, 4065-4074.

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Immunohistochemistry for Neural Markers

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The following antibodies were used: anti–phospho-histone H2A.X (Ser¹³⁹) antibody, clone JBW301, EMD Millipore, 05-636, 1:500; NeuN, Synaptic Systems, 266 004, 1:1000; CaMKII-α (6G9) mouse monoclonal antibody (mAb), Cell Signaling Technology, 50049S, 1:200; NEUROD1 polyclonal antibody, Proteintech, 12081-1-AP, 1:200; anti-GFP antibody, Abcam, ab13970, 1:500; Iba1, Synaptic Systems, 234 004, 1:1000; anti-GFAP antibody, Abcam, ab53554, 1:500; recombinant anti-Olig2 antibody (EPR2673), Abcam, ab109186, 1:500; RFP antibody preadsorbed, Fisher Scientific, 600-401-379, 1:200; NFκB p65 (D14E12) XP rabbit mAb, Cell Signaling Technology, 8242S, 1:500; NFκB p65 polyclonal antibody, Invitrogen, 51-0500, 1:100; anti–MHC class II (I-A/I-E), clone M5/114, EMD Millipore, MABF33, 1:500; anti-nestin antibody (rat 401), Abcam, ab6142, 1:1000; anti-C1q antibody (4.8), Abcam, ab182451, 1:500; synapsin 2 antibody (guinea pig), Synaptic Systems, 106 004, 1:500; VGlut1 (rabbit), Synaptic Systems, 135 303, 1:500; purified anti-mouse/rat β-amyloid antibody, BioLegend, 805801, 1:500; phospho-Tau (Thr¹⁸¹) (D9F4G) rabbit mAb, Cell Signaling Technology, 12885S, 1:500.

Welch G.M., Boix C.A., Schmauch E., Davila-Velderrain J., Victor M.B., Dileep V., Bozzelli P.L., Su Q., Cheng J.D., Lee A., Leary N.S., Pfenning A.R., Kellis M, & Tsai L.H. (2022). Neurons burdened by DNA double-strand breaks incite microglia activation through antiviral-like signaling in neurodegeneration. Science Advances, 8(39), eabo4662.

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Western Blot Analysis of Smad and NF-κB Signaling

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Cells were lysed with RIPA Lysis and Extraction Buffer (Thermo Scientific cat.# PI89900) buffer with protease/phosphatase inhibitor cocktail. The lysate was centrifuged at 12,000×g at 4°C for 15 min, boiled with Laemmli buffer for 7 min at 95°C, and transferred to PVDF membranes (Millipore). After blocking, membranes were incubated with primary antibody at 4°C overnight followed by incubation with corresponding secondary HRP-linked antibody. The following antibodies were used for western blotting: Smad1 (D59D7) XP Rabbit mAb (Cell Signaling Technology cat.# 6944), Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad9 (Ser465/467) (Cell Signaling Technology cat.# 13,820P), NF-κB p65 (D14E12) XP Rabbit mAb (Cell Signaling Technology cat.# 8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (Cell Signaling Technology cat.# 3033), Anti-GAPDH antibody EPR16884 Loading Control (Abcam cat.# ab181603).

Xiong L., Zhevlakova I., West X.Z., Gao D., Murtazina R., Horak A., Brown J.M., Molokotina I., Podrez E.A, & Byzova T.V. (2024). TLR2 regulates hair follicle cycle and regeneration via BMP signaling. eLife, 12, RP89335.

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NOS1 Deficiency in Mice and LPS-induced Inflammation

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6 to 8 weeks old WT and NOS1^−/− C57BL/6 J mice were obtained from the Jackson Laboratory. The study was approved by the ethics board of Chongqing University of Posts and Telecommunications (CQUPT) (No. CA2019-01). Animal handling and experiments were conducted in accordance with the policies of the Animal Care Facility of CQUPT. LPS from Escherichia coli O111:B4 was obtained from Sigma. IL4 was purchased from PeproTech. The RNeasy Mini Kit was purchased from Qiagen, and the High Capacity cDNA Reverse Transcription Kit and Fast SYBR^® Green Master Mix were obtained from Applied Biosystems. The STAT6 (C9) mouse monoclonal antibody (mAb) was purchased from Santa Cruz Biotechnology. β-Actin (8H10D10) mouse mAb, NF-κB p65 (D14E12) XP^® rabbit mAb, iNOS (D6B6S) rabbit mAb, PPARγ (C26H12) rabbit mAb, and Phospho-Stat6 (Tyr641) (D8S9Y) rabbit mAb were obtained from Cell Signaling Technology. The SOCS1 rabbit antibody (ab62584) was purchased from Abcam.

Chang P., Gao H., Sun Q., He X, & Huang F. (2021). Transcriptome profiling reveals new insights into the roles of neuronal nitric oxide synthase on macrophage polarization towards classically activated phenotype. PLoS ONE, 16(9), e0257908.

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BAFF Pathway Activation in B Cells

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B cells were isolated from mouse spleen and 6 million B cells/sample were treated with 5 nM BAFF 3-mer, 60-mer, or left untreated for 3 hours or 24 hours. Alternatively, B cells were treated with 30 nM of the control antibody or the mBaffR-Fc and BAFF proteins. After the treatments, the cells were washed, and total cellular protein was extracted using 1x RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitors (SigmaAldrich). The extracted protein was analyzed using 4-12% Tris-Glycine SDS-PAGE. The primary antibodies used are Phospho-NF-κB p65 (Ser536) Antibody (#3031), NF-κB p65 (D14E12) XP^® Rabbit mAb (#8242), NF-κB2 p100/p52 Antibody (#4882), and β-Actin Antibody (#4967) from Cell Signaling Technology, OxPhos Rodent WB Antibody Cocktail (#45-8099) from ThermoFisher Scientific, the IRDye-conjugated secondary antibodies were from LI-COR, and the blots were detected, and band intensities were quantified using LI-COR’s Image Studio software version 5.2.5.

van der Vlugt-Bergmans C.J., Meeuwsen P.J., Voragen A.G, & van Ooyen A.J. (2000). Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme. Applied and Environmental Microbiology, 66(1), 36-41.

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