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|  | ANTIBODIES TO PROTEINS INVOLVED NF-KAPPA B SIGNALING
Over 20 years ago, in a search for factors that bind immunoglobulin heavy chain and kappa light chain gene enhancers, Ranjan Sen and David Baltimore identified the nuclear factor, NF-kappa B (nuclear factor that binds the kappa immunoglobulin light chain gene enhancer). At the time of its discovery, it was hypothesized that this factor would represent a cell specific transcription factor required for B-cell immunoglobulin (Ig) gene expression (Sen & Baltimore, 1986); however soon it became evident that the predicted cell-specific function of NF-kappa B would reach beyond B-cells and Ig expression. Instead, NF-kappa B would encompass a much wider functional arena that involved the activation of a number of pleiotropic transcriptional programs (Lenardo & Baltimore, 1989).
Classically, NF-kappa B is described as a transcription factor that reprograms gene expression of the immune system in response to injury, infection, and stress. The NF-kappa B transcription factor exists as a dimer composed of pairs of monomeric subunits that are part of the Rel Family of proteins (Perkins & Gilmore, 2006). The Rel family of proteins and their related sequences were first identified in an oncogene derived from the avian reticuloendotheliosis retrovirus (Stephens, Rice, Hiebsch, Bose, Jr., & Gilden, 1983). The members of this family include NF-kappa B1/p50, NF-kappa B2/p52, RelA/p65, c-Rel, and RelB.
The combinatorial nature of the NF-kappa B transcription factor provides a level of regulation that allows it to mediate an array of pleiotropic responses to a diverse set of stimuli. Another level of NF-kappa B regulation is seen in the form of associated inhibitor proteins that interact with NF-kappa B to inhibit its DNA binding activity. These inhibitor proteins are members of the I kappa B family of proteins and include I kappa B-beta, I kappa B-delta/NF-kappa B2/p100, I kappa B-gamma/NF-kappa B/p105, I kappa B-epsilon, I kappa B-zeta, and Bcl3. Association with the inhibitory I kappa B proteins sequesters NF-kappa B in the cytoplasm to regulate its activity. The phosphorylation of I kappa B by the I kappa B kinase complex (IKK) results in the ubiquitination and degradation of I kappa B and the release and translocation of NF-kappa B to the nucleus where it can activate a vast number of genes. The regulatory kinase, IKK, is composed of two catalytic subunits, IKK-alpha and IKK-beta, and a regulatory subunit IKK-gamma/NEMO. Non-canonical regulatory mechanisms that do not involve the regulation NF-kappa B by the I kappa B proteins also exist. Such mechanisms include posttranslational modification, such as phosphorylation, of the NF-kappa B members (Neumann & Naumann, 2007).
Due to its combinatorial nature, its ubiquitous expression, and its ability to mediate pleiotropic responses, NF-kappa B has become one of the most studied transcription factors. Recently, the dysregulation of NF-kappa B has been associated with a multitude of diseases and is being considered as a potential target of pharmacological intervention (Kumar, Takada, Boriek, & Aggarwal, 2004). These points illustrate that our understanding of NF-kappa B is far from complete and that it will to continue as a subject of intense study.
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Detection of Human and Mouse RelA/p65 by Western Blot (human & mouse) and Immunoprecipitation (human).
Samples: Whole cell lysate from HeLa (H; 50 mcg for WB; 1 mg for IP, 20% of IP loaded), 293T (T; 50 mcg), and mouse NIH3T3 (M; 50 mcg) cells.
Antibodies: Affinity purified rabbit anti-RelA/p65 antibody A301-824A used for Western Blot at 0.4 mcg/ml (A) and 1 mcg/ml (B) and used for Immunoprecipitation at 3 mcg/mg lysate. RelA/p65 was also immunoprecipitated by rabbit anti-RelA/p65 antibody A301-823A, which recognizes an upstream epitope. For blotting immunoprecipitated RelA/p65, the ReliaBLOT® Reagents and Procedures (Cat. No. WB120) were used.
Detection: Chemiluminescence with exposure times of 30 seconds (A and B).
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Detection of Human NF-kappaB1 by Western Blot and Immunoprecipitation.
Samples: Whole cell lysate from HeLa (H; 50 mcg) and Jurkat (J; 50 mcg for Western Blot; 1 mg for IP, 20% of IP loaded) cells.
Antibodies: Affinity purified rabbit anti-NF-kappaB1 antibody A301-820A used for WB at 0.04 mcg/ml (A) and 0.4 mcg/ml (B) and used for IP at 3 mcg/mg lysate. NF-kappaB1 was also immunoprecipitated by rabbit anti-NF-kappaB1 antibody A301-819A, which recognizes an upstream epitope. For blotting immunoprecipitated NF-kappaB1, the ReliaBLOT® Reagents and Procedures (Cat. No. WB120) were used.
Detection: Chemiluminescence with exposure times of 1 minute (A) and 3 seconds (B). |
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Reference List
Kumar, A., Takada, Y., Boriek, A. M., & Aggarwal, B. B. (2004). Nuclear factor-kappaB: its role in health and disease. J. Mol. Med., 82, 434-448.
Lenardo, M. J. & Baltimore, D. (1989). NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell., 58, 227-229.
Neumann, M. & Naumann, M. (2007). Beyond IkappaBs: alternative regulation of NF-kappaB activity. FASEB J., 21, 2642-2654.
Perkins, N. D. & Gilmore, T. D. (2006). Good cop, bad cop: the different faces of NF-kappaB. Cell Death. Differ., 13, 759-772.
Sen, R. & Baltimore, D. (1986). Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell., 46, 705-716.
Stephens, R. M., Rice, N. R., Hiebsch, R. R., Bose, H. R., Jr., & Gilden, R. V. (1983). Nucleotide sequence of v-rel: the oncogene of reticuloendotheliosis virus. Proc. Natl. Acad. Sci. U.S.A., 80, 6229-6233.
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