RelA/NFkB p65 Antibody (532301) [Unconjugated]

Western Blot shows lysates of HeLa human cervical epithelial carcinoma cell line and K562 human chronic myelogenous leukemia cell line. PVDF membrane was probed with 2 µg/ml of Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Western Blot shows lysates of BaF3 mouse pro-B cell line. PVDF membrane was probed with 2 µg/ml of Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Western blot shows lysates of C6 rat glioma cell line. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

RelA/NF kappa B p65 was detected in immersion fixed K562 human chronic myelogenous leukemia cell line using Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

HeLa human cervical epithelial carcinoma cell line was stained with Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078, filled histogram) or isotype control antibody (Catalog # MAB0041, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). To facilitate intracellular staining, cells were fixed with paraformadehyde and permeabilized with methanol.

Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and RelA/NF kappa B p65 knockout HeLa cell line (KO). PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Non-classical and intermediate monocytes express high levels of NF-kappa B (p65) and membrane-bound IL-1 alpha .a Western blot analysis of total p65 and GAPDH protein levels in the three monocyte subsets. b Quantification of Western blot data shown in (a): p65 protein level was normalized to GAPDH (loading control) and expressed as a fold change with respect to CL subset. The data represent the means ± SD; n = 3. c Relative levels of phosphorylated-p65 (p-p65), measured by flow cytometry. Each line represents one donor; n = 7. d IL-1 alpha secretion by the three monocyte subsets, measured by Luminex assay. The data represent the means ± SD; n = 3. e–f Relative expression levels of membrane-bound IL-1 alpha (e) and cytoplasmic IL-1 alpha (f), analyzed by flow cytometry. Each line represents one donor; n = 9. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CL: classical, ITM: intermediate, NC: non-classical, MFI: median fluorescence intensity Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41419-018-0327-1), licensed under a CC-BY license. Not internally tested by R&D Systems.

Tumor‐derived EVs activate the NF kappa B pathway. Primary CD14+ monocytes were incubated with TEVs isolated from conditioned PCI‐1 supernatants. (A) Lysates were generated at different timepoints of incubation and tested for I kappa B and phosphorylated P‐I kappa B. Tubulin was used as a loading control. (B) Primary monocytes were incubated with complete PCI‐1 supernatant (SN), in TEV‐depleted SN (depl.) or with isolated TEV for 120 min, nuclei were isolated and the translocation of NF kappa B‐p65 was tested with an immunoblot. Actin was used as a loading control, and the cytoplasmic fraction (p65/cyto) was used to confirm the specificity of the translocation. (C) Monocytes were incubated as in (B), and the binding capacity of NF kappa B p50 to the DNA was quantified with a NF kappa B binding assay kit. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29601673), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu, Mu, Rt
• Applications: WB, CyTOF-ready, Flow, ICC/IF, KO
• Clone: 532301
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

RelA/NFkB p65 Antibody (532301) [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human RelA/NF kappa B p65 isoform 1
  Asn456-Ser551
  Accession # Q04206
• Specificity: Detects human, mouse, and rat RelA/NF kappa B p65 in Western blots.
• Source: N/A
• Isotype: IgG2b
• Clonality: Monoclonal
• Host: Mouse
• Gene: RELA
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • CyTOF-ready
  • Immunocytochemistry 8-25 ug/mL
  • Intracellular Staining by Flow Cytometry 2.5 ug/10^6 cells
  • Knockout Validated
  • Western Blot 2 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. See Certificate of Analysis for details.
  *Small pack size (-SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for RelA/NFkB p65 Antibody (532301) [Unconjugated]

• MGC131774
• nf kb p65
• NFkB p65
• NF-kB p65
• NFKB3
• NFKB3v-rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor ofkappa light polypeptide gene enhancer in B-cells 3 (p65))
• Nuclear factor NF-kappa-B p65 subunit
• Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3transcription factor p65
• p65
• p65RelA
• rela p65
• RelA
• v-rel reticuloendotheliosis viral oncogene homolog A (avian)
• v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappalight polypeptide gene enhancer in B-cells 3, p65

Background

RelA p65 (v-rel reticuloendotheliosis viral oncogene homolog A) is a 65 kDa member of the NF kappa B family of nuclear transcription factors. Dimers of p65 with the p50 subunit are the most common form of the NF kappa B transcription factor, but dimers with it or other family members can also occur. Upon activation, RelA p65 forms an heterotetramer and moves into the nucleus where it binds to specific DNA sequences. An alternatively spliced isoform that lacks amino acids (aa) 222‑231 (p65 delta ) does not bind DNA. Over the sequence used as an immunogen, human RelA p65 shares 96% and 98% aa identity with mouse and rat RelA p65, respectively. This portion includes one of eight potential ser/thr phosphorylation sites, two acetylation sites, and most of the Rel homology domain that interacts with I kappa B inhibitors.

 

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for RelA/NFkB p65 Antibody (MAB5078)(12)

Publications using MAB5078

• Suehiro, JI;Kimura, T;Fukutomi, T;Naito, H;Kanki, Y;Wada, Y;Kubota, Y;Takakura, N;Sakurai, H; Endothelial specific LAT1 ablation normalizes tumor vasculature JCI insight 2024-08-20 [PMID: 39163136] (Western Blot, Human)
• Meah, A;Vedarethinam, V;Bronstein, R;Gujarati, N;Jain, T;Mallipattu, SK;Li, Y;Wang, J; Single-Cell Spatial MIST for Versatile, Scalable Detection of Protein Markers Biosensors 2023-08-27 [PMID: 37754086] (IHC, Mouse)
• Brockmueller, A;Girisa, S;Motallebi, M;Kunnumakkara, AB;Shakibaei, M; Calebin A targets the HIF-1?/NF-?B pathway to suppress colorectal cancer cell migration Frontiers in pharmacology 2023-07-31 [PMID: 37583906] (Immunoprecipitation, Human)
• Festa J, Hussain A, Hackney A et al. Elderberry extract improves molecular markers of endothelial dysfunction linked to atherosclerosis Food Science & Nutrition 2023-07-01 [PMID: 37457144]
• A Brockmuell, S Girisa, AB Kunnumakka, M Shakibaei Resveratrol Modulates Chemosensitisation to 5-FU via beta1-Integrin/HIF-1alpha Axis in CRC Tumor Microenvironment International Journal of Molecular Sciences, 2023-03-05;24(5):. 3/5/2023 [PMID: 36902421]
• A Brockmuell, AL Mueller, P Shayan, M Shakibaei beta1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment Frontiers in Pharmacology, 2022-09-02;13(0):978625. 2022-09-02 [PMID: 36120305] (ICC/IF, Human)
• Xu X, Qiao D, Pan L et al. RELA 8-Oxoguanine DNA Glycosylase1 Is an Epigenetic Regulatory Complex Coordinating the Hexosamine Biosynthetic Pathway in RSV Infection Cells 7/15/2022 [PMID: 35883652]
• A Makowska, N Lelabi, C Nothbaum, L Shen, P Busson, TTB Tran, M Eble, U Kontny Radiotherapy Combined with PD-1 Inhibition Increases NK Cell Cytotoxicity towards Nasopharyngeal Carcinoma Cells Cells, 2021-09-17;10(9):. 2021-09-17 [PMID: 34572108] (Flow Cytometry, Human)
• P Wohlrab, L Soto-Gonza, T Benesch, MP Winter, IM Lang, K Markstalle, V Tretter, KU Klein Intermittent Hypoxia Activates Duration-Dependent Protective and Injurious Mechanisms in Mouse Lung Endothelial Cells Front Physiol, 2018-12-06;9(0):1754. 2018-12-06 [PMID: 30574096] (Western Blot, Mouse)
• Kathrin Gärtner, Christina Battke, Judith Dünzkofer, Corinna Hüls, Bettina von Neubeck, Mar‐ kus Kellner et al. Tumor‐derived extracellular vesicles activate primary monocytes Cancer Medicine 2018-05-01 [PMID: 29601673]
• SM Ong, E Hadadi, TM Dang, WH Yeap, CT Tan, TP Ng, A Larbi, SC Wong The pro-inflammatory phenotype of the human non-classical monocyte subset is attributed to senescence Cell Death Dis, 2018-02-15;9(3):266. 2018-02-15 [PMID: 29449647] (Western Blot, Human)
• Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor treated CML stem cells Blood, 2016-09-12;0(0):. 9/12/2016 [PMID: 27621307]

Bioinformatics

• Gene Symbol: RELA
• Uniprot:
  • Q04206()