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8214
PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet
Primary Antibodies
Antibody Duet

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet #8214

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PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 1

Flow cytometric analysis of HeLa cells, untreated (blue) or TNF-α-treated (green), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb compared to a nonspecific negative control antibody (red).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 2

CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across LAMC2, a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IL-8, a known target gene of NFκB (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 4

Flow cytometric analysis of HeLa cells using NF-κB p65 (D14E12) XP® Rabbit mAb (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 5

Confocal immunofluorescent analysis of HeLa cells, serum starved (left) or TNF-α treated (#8902 at 20 ng/ml for 20 min, right), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® phalloidin 555 (red).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 6

CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 1 (upper), including LAMC2 (lower), a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 7

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by Real-Time PCR using SimpleChIP® Human IκBα Promoter Primers #5552, human IL-8 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 8

Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or TNF-α treated (#2169, 20 ng/ml for 5 minutes), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) or NF-κB p65 Antibody #3034 (lower).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 9

CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human LAMC2 upstream primers, and human ITM2A upstream primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 10

Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) and NF-κB p65 (C22B4) Rabbit mAb #4764 (lower).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 11

Confocal immunofluorescent analysis of HT-1080 cells, untreated (left) or treated with hTNF-α #8902 (20 ng/ml, 20 min) (right), using NF-κB p65 (D14E12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 12

Immunohistochemical analysis using NF-κB p65 (D14E12) XP® Rabbit mAb on SignalSlide® NF-κB p65 IHC Controls #12873 (paraffin-embedded HCT116 cells, untreated (left) or treated with hTNF-α #8902 (right)).

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 13

Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.

PhosphoPlus® NF-κB p65/RelA (Ser536) Antibody Duet: Image 14

Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.

To Purchase # 8214S
Product # Size Price
8214S
1 Kit $ 537

Product Description

PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.

Background

Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).

  1. Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
  2. Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
  3. Haskill, S. et al. (1991) Cell 65, 1281-9.
  4. Thompson, J.E. et al. (1995) Cell 80, 573-82.
  5. Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.
  8. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  9. Senftleben, U. et al. (2001) Science 293, 1495-9.
  10. Coope, H.J. et al. (2002) EMBO J 21, 5375-85.
  11. Xiao, G. et al. (2001) Mol Cell 7, 401-9.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus is a trademark of Cell Signaling Technology, Inc.