Antibodies to Proteins Involved in NF-kappaB Signaling

Over 20 years ago factors that bind immunoglobulin heavy chain and kappa light chain gene enhancers had been searched for.
Ranjan Sen and David Baltimore identified the nuclear factor, NF-kappaB (nuclear factor that binds the kappa immunoglobulin light chain gene enhancer). At the time of its discovery, NF-kappaB was hypothesized to represent a cell specific transcription factor required for B-cell immunoglobulin (Ig) gene expression; instead it was soon determined that NF-kappaB would encompass a much wider functional arena that involved the activation of a number of pleiotropic transcriptional programs.

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.

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).

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).

Product Links

	Applications
Antibody	Western Blot	Immunoprecipitation
I kappa B-beta	A301-828A	A301-828A
IKK-beta	A301-827A	A301-827A
NF-kappaB1	A301-820A	A301-819A A301-820A
NF-kappaB2	A301-821A A301-822A	A301-821A A301-822A
RelA/p65	A301-824A	A301-823A A301-824A
c-Rel	A301-826A	A301-825A A301-826A
CKII alpha	A300-196A A300-197A	A300-198A

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.