Phospho-NF-κB p65 (Ser468) Ant

3039:

Immunoprecipitation , Western Blotting

Phospho-NF-kappaB p65 (Ser468) Antibody detects NF-kappaB p65 only when phosphorylated at serine 468.

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser468 of human NF-kappaB p65. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western blot analysis of extracts from HeLa cells treated for 5 minutes with TNF-alpha #2169 (20 ng/ml), Calyculin A #9902 (50 nM), or both compounds, using Phospho-NF-kappaB p65 (Ser468) Antibody (top) or NF-kappaB p65 Antibody #3034 (bottom).

Transcription factors of the nuclear factor κ B (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 is then translocated to the nucleus (9-11).

PMA-induced NF-kappaB transcriptional activity is dependent on the region between amino acids 442 and 470, suggesting a role for one or more of the potential phosphorylation sites (Ser457, Thr458, Thr464, or Ser468) in this region (12). T-cell costimulation and Calyculin A have both been shown to increase Ser468 phosphorylation (13, 14). IKKβ (but not IKKα) and GSK-3β both target this site (14, 15), which appears to have a negative regulatory role not involving inhibition of nuclear translocation after TNFα or IL-1β stimulation (15).

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.
12. Schmitz, M.L. et al. (1995) J Biol Chem 270, 15576-84.
13. Mattioli, I. et al. (2004) Blood 104, 3302-4.
14. Buss, H. et al. (2004) J Biol Chem 279, 49571-4.
15. Schwabe, R.F. and Sakurai, H. (2005) FASEB J 19, 1758-60.

• Mattioli, I. et al. (2004) Blood 104, 3302-4. Applications: Western Blotting
• Buss, H. et al. (2004) J Biol Chem 279, 49571-4. Applications: Western Blotting

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• 4882 NF-κB2 p100/p52 Antibody
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• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

For Research Use Only. Not For Use In Diagnostic Procedures.