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Transcription Factor NFκB

The transcription factor NFkB (NF kappa B, NF-kB, or nuclear factor kB) is widely studied due to its implication in the regulation of genes that control inflammation, cell proliferation and cell survival. NFkB can be activated by exposure of cells to stimuli from cytokines such as TNF (tumor necrosis factor) or IL-1 (interleukin-1), free radicals, ultraviolet irradiation, stress and bacterial & viral agents1-3. Aberrant activation of NFkB is observed in many human cancers.

Diagram of NFkB activation.
Diagram of NFκB activation.
 

NFkB is comprised of homo- or heterodimers of different subunits. These subunits are members of the structurally related Rel family of transcription factors. Five different Rel proteins (also called Rel/NFkB proteins) have been identified: p50 (NF-kB1), p52 (NF-kB2), p65 (RelA), RelB and c-Rel4. The p50/p65 heterodimer is the major Rel/NFkB complex found in most cells. Rel proteins contain an N-terminal region called the Rel Homology Domain (RHD) that is conserved among the family members and is responsible for DNA-binding and dimerization.

Two groups of Rel/NFkB proteins are typically described: RelA, RelB and c-Rel contain a transactivation domain (TD) in their C-termini, which is required for the transport of active NFkB complexes into the nucleus. In contrast, subunits p50 and p52 do not contain transactivation domains; they are unable to transactivate on their own and must form heterodimers with RelA, RelB or c-Rel. Homodimers of p50 and p52 were even reported to repress kB site-dependent transcription in vivo, possibly by competing with other transcriptionally active dimers (e.g. p50/p65) for DNA binding5. NF-kB1 and NF-kB2 are synthesized as the large precursors p105 and p100, respectively. The p105 precursor undergoes constitutive processing to generate the mature subunit p50, while the processing of p100 is tightly regulated to generate the mature subunit p526, 7.

Inactive NFkB dimers are sequestered in the cytoplasm of cells by the IκB family of inhibitory proteins. IkB proteins are a family of related proteins containing an N-terminal regulatory domain followed by six or more ankyrin repeats. These ankyrin repeats interact with the RHD of NFkB proteins, masking their nuclear localization signal and preventing translocation8. The IkB family includes: IkBa, IkBb, IkBe, IkBg and Bcl-3.

Activation of NFkB can occur internally by acetylation of p65 (RelA) usually at lysines 218, 221 or 310. Acetylated NFkB is active and resistant to the inhibitory effects of IkB. Activation of NFkB by external inducers requires the phosphorylation and subsequent degradation of IkB proteins. Phosphorylation of IkB proteins by IkB kinases (IKK) at two serine residues, Ser32 & Ser36, located in the IkB regulatory domain leads to ubiquitination of the inhibitor IkB molecules, which are then degraded by the proteasome. IKK is composed of a heterodimer of catalytic IKKa and IkBb and a regulatory protein NEMO (NFkB Essential Modulator). The activated NFkB complex translocates to the nucleus and binds DNA to regulate gene expression.

In addition to activating genes for inflammation, cell survival and cell proliferation, NFkB also turns on expression of its own repressor, IkBa. The newly synthesized IkBa then re-inhibits NFkB, creating an auto feedback loop. This results in oscillating levels of NFkB activity9.

References

  1. Gilmore, T.D. (2006) Oncogene 25: 6680-6684.
  2. Brasier, A.R. (2006) Cardiovasc. Toxicol 6: 111-130.
  3. Perkins, N.D. (2007) Nat. Rev. Mol. Cell Biol. 8: 49-62.
  4. Nabel, G.J., Verma, I.M. (1993) Genes Dev. 7: 2063.
  5. Lernbecher et al. (1993) Nature 365: 767-770.
  6. Karin, M., Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18: 621-663.
  7. Senftleben et al. (2001) Science 293: 1495-1499.
  8. Jacobs, M.D., Harrison, S.C. (1998) Cell 95: 749-758.
  9. Nelson et al. (2004) Science 306: 704-708.

 

Active Motif Tools to Analyze NFκB Function

Luminex® Transcription Factor Multiplex Assays

Active Motif has partnered with Luminex®, the industry leader for multiplexing, to develop Transcription Factor Multiplex Assays for studies of AP-1 and NFκB transcription factor families. The assays are designed for use with either the Luminex® 200™ or MAGPIX® instruments for multiplexing of analyte-specific DNA binding events. These highly sensitive assays require small sample input and can be adapted for high-throughput analysis, enabling researchers to collect more data at a faster rate using less sample than afforded by traditional methods. The Transcription Factor Multiplex assays work as pull-down reactions where lysates are combined with a biotinylated consensus binding sequence for the transcription factor family of interest. Antibodies specific for each transcription factor analyte are conjugated to fluorescent-labeled magnetic beads and used to capture the transcription factor-bound DNA. The fluorescent label of the bead is specific to each analyte, enabling multiplexing of analytes within the same sample. Streptavidin-phycoerythrin (SA-PE) is used to bind the biotinylated oligo and provide a readout to determine the magnitude of signal that is proportional to the amount of transcription factor binding.

For more information about Luminex Transcription Factor Multiplex Assays and available analytes, please visit Luminex® Transcription Factor Multiplex Assays.

TransAM® transcription factor DNA-binding ELISAs

Active Motif‘s TransAM Kits are ELISA-based, DNA-binding assays that facilitate the study of transcription factor activation in mammalian tissue and cell culture extracts. The TransAM method provides quantitative results in less than 5 hours, eliminates the use of radioactivity or the need to run gels and is up to 100-fold more sensitive than classic electrophoretic mobility shift assay (EMSA), or gelshift techniques.

TransAM NFκB Kits use a 96-stripwell plate to which a double-stranded oligonucleotide containing the NFkB-binding site has been immobilized. When nuclear or whole-cell extracts are added to the plate, activated NFkB binds to its consensus sequence on the plate-bound oligonucleotide. Next, a primary antibody for either p50, p65, p52, RelB or c-Rel is added. This is detected by incubation with a secondary HRP-conjugated antibody and developing reagent. The colorimetric TransAM format is measured using a spectrophotometer, while the more sensitive chemiluminescent TransAM Kits use a luminometer. The readout provides a quantitative measure of activated NFkB. Recombinant proteins for p65 and p50 are also available separately for use as a protein standard in the TransAM Kits.

To learn more, please see the information on our TransAM transcription factor ELISAs.

Gelshift™ Chemiluminescent EMSA

While Active Motif believes that its TransAM method is far superior to EMSA, we still offer the Gelshift Chemiluminescent EMSA Kit as a simple, non-radioactive option for performing EMSA to study the DNA binding activity of transcription factors, including NFkB.

For more information, please read about our EMSA related products on our Gelshift assay page.

LigandLink™ for fluorescent labeling and protein localization studies

LigandLink enables you to label your protein of interest in living cells. Simply express the protein as a LigandLink fusion and add one of the cell permeable LigandLink labels to the medium. Monitor protein localization and protein:protein interactions with either red or green fluorescence.

LigandLink vectors are available with an empty MCS, or with an NFκB p65 fusion. (Other transcription factor fusions are also available.) LigandLink labels include fluorescein or hexachlorofluorescein. To learn more, visit the LigandLink information pages.

FACE™ cell-based ELISAs

Fast, Activated Cell-based ELISA (FACE) Kits provide a cell-based method to monitor proteins activated by phosphorylation. In this in-cell Western method, cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are fixed to preserve activation-specific protein modifications, such as phosphorylation. Each well is then incubated with a primary antibody specific for the activated protein of interest, such as total NFkB, phospho-NFkB Ser468, or phospho-NFkB Ser536. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides a colorimetric or chemiluminescent readout that is quantitative and reproducible. (In addition to NFkB, FACE Kits for over 20 other phospho-proteins are available.)

For more information, please visit the pages that describe our FACE cell-based ELISAs.

FunctionELISA™ IκBα for analysis of IκBα phosphorylation

Active Motif‘s FunctionELISA Kits offer a rapid method to monitor changes in signal pathway proteins, such as IkBa. FunctionELISA IκBα utilizes the Sandwich ELISA technique to capture and quantifiably measure the amount of phosphorylated IkBa present in a sample. This kit has the ability to assay both cell and tissue samples without the need to run, blot and develop gels. (FunctionELISA Kits for TRAIL and cytochrome c are also available.)

To learn more, visit the FunctionELISA information pages.

ChIP-IT® Express for binding site analysis

Active Motif offers ChIP-IT Express Kits for chromatin immunoprecipitation (ChIP) to study transcription factor binding sites. ChIP-IT Express utilizes magnetic protein G beads, which is a large improvement over agarose bead-based ChIP, and is available in either a sonication or enzymatic format for chromatin preparation. Active Motif also offers a large number of ChIP-validated antibodies and Ready-to-ChIP Chromatin, which help you perform ChIP successfully.

To learn more about ChIP-IT Express and many other products for chromatin IP, please visit our ChIP-IT Express description.

Universal Magnetic Co-IP to detect protein interactions

Active Motif‘s Universal Magnetic Co-IP Kit enables co-immunoprecipitation for both nuclear and whole-cell complexes. It takes advantage of the same magnetic bead-based protocol utilized in ChIP-IT Express to speed and simplify the IP and wash steps, while greatly reducing background.

For additional information, please visit the page on our Universal Magnetic Co-IP Kit.

SUMOlink™ for SUMOylation studies

Active Motif‘s SUMOlink Kits are used to study SUMOylation, a post-translational modification that affects many biological processes. SUMO is a small ubiquitin-like modifier that shares different degrees of sequence identity with ubiquitin. SUMOlink provides a simple method for generating and detecting SUMOylated proteins in vitro to study either SUMO-1 or SUMO-2 & SUMO-3 activity. The kits provide all the reagents required for the SUMOylation including positive and negative controls to help ensure success.

For more information, visit the SUMOlink information pages.

Recombinant proteins

Active Motif carries recombinant proteins for many transcription factors and cell signaling-related proteins, including NFκB p65 and NFκB p50 proteins that have been validated for use as protein standards in TransAM Transcription Factor ELISAs.

To see our complete list of recombinant proteins, please click here.

Antibodies

Check out Active Motif‘s broad line of mono- and polyclonal antibodies directed against Rel/NFkB, IkB and IKK proteins among others. Active Motif‘s antibodies are highly characterized, and many have been validated for use in ChIP, Western blot or EMSA. To see all of our antibodies, please click here.

Cell extracts

Active Motif offers high-quality nuclear, cytoplasmic and whole-cell extracts from a variety of cell types and tissue sources. Many of our extracts are prepared from cells that have been treated to specifically induce activation of hard-to-detect transcription factors. Our ready-to-use extracts are ideal as positive controls in a variety of applications, including TransAM, gelshift and Western blots.

We have a wide selection of extracts to choose from, many of which are ideal controls for NFkB studies. To see our list of available extracts, please click here.