Product Pathways - NF-kB Signaling
NF-κB p65 Antibody Sampler Kit #4767
|4767S||1 Kit (5 x 40 µl)||---||In Stock||---|
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|Kit Includes||Quantity||Applications||Reactivity||Homology†||MW (kDa)||Isotype|
|Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033||40 µl||W, IP, IF-IC, F||H, M, R, Hm, Mk, Pg||Dg||65||Rabbit IgG|
|Acetyl-NF-κB p65 (Lys310) Antibody #3045||40 µl||W, IP||H, M||R, Mk, B, Dg||65||Rabbit|
|NF-κB p65 (L8F6) Mouse mAb #6956||40 µl||W, IP, IHC-P, IF-IC, F, ChIP||H, M, R, Hm, Mk, Mi, B, Dg, Pg||65||Mouse IgG2b|
|NF-κB p65 (D14E12) XP® Rabbit mAb #8242||40 µl||W, IP, IHC-P, IF-IC, F, ChIP||H, M, R, Hm, Mk, Dg||65||Rabbit IgG|
|Phospho-NF-κB p65 (Ser468) Antibody #3039||40 ml||W, IP||H, M, R||65||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
|Anti-mouse IgG, HRP-linked Antibody #7076||100 µl||Horse|
†Species predicted to react based on 100% sequence homology.
Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, IHC-P=Immunohistochemistry (Paraffin), ChIP=Chromatin IP
Reactivity Key: H=Human, M=Mouse, R=Rat, Hm=Hamster, Mk=Monkey, Pg=Pig, Mi=Mink, B=Bovine, Dg=Dog
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 #3033 (upper) or NF-κB p65 Antibody #3034 (lower).
Western blot analysis of extracts from HeLa cells treated for 5 minutes with TNF-α #2169 (20 ng/ml), Calyculin A #9902 (50 nM), or both compounds, using Phospho-NF-kB p65 (Ser468) Antibody #3039 (top) or NF-kB p65 Antibody #3034 (bottom).
Western blot analysis of extracts from HeLa cells, mock transfected or transfected with NF-κB or NF-κB plus CBP, using Acetyl-NF-κB (Lys310) Antibody #3045. Western blots were competed with acetylated peptide (middle) or non-acetylated peptide (right).
Western blot analysis of extracts from various cell lines using NF-κB p65 (L8F6) Mouse mAb #6956.
The NF-κB p65 Antibody Sampler Kit contains reagents to examine NF-κB p65/RelA phosphorylation at Ser468 and Ser536; acetylation at Lys310; and total p65 levels.
Specificity / Sensitivity
The total NF-κB p65 antibodies recognize endogenous levels of p65 regardless of post-translational modification state such as phosphorylation or acetylation. The phospho-NF-κB p65 antibodies recognize endogenous levels of p65 only when phosphorylated at their indicated target residues. The acetyl-NF-κB p65 (Lys310) antibody recognizes transfected levels of p65 only when acetylated at Lys310.
Source / Purification
The NF-κB p65 (L8F6) Mouse mAb was produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human NF-κB p65. The NF-κB p65 Antibody was produced by immunizing rabbits with a synthetic peptide corresponding to amino acids surrounding Glu498 of human NF-κB p65. The phospho-specific antibodies were produced by immunizing rabbits with synthetic phosphopeptides corresponding to amino acids surrounding the indicated target residues of human NF-κB p65. The Acetyl-NF-κB p65 (Lys310) Antibody was prepared by immunizing rabbits with a synthetic acetylated peptide corresponding to residues surrounding Lys310 of human NF-κB p65. Polyclonal antibodies were purified by protein A and peptide affinity chromatography.
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 translocates to the nucleus (9-11).
RelA/p65 is a subunit of the NF-κB transcription complex, which plays a crucial role in inflammatory and immune responses. The complex is composed of various homodimeric and heterodimeric Rel family member combinations, the activity of which is modulated by post-translational modifications including phosphorylation and acetylation. p65 phosphorylation by PKA and/or MSK1 at Ser276 allows for increased interaction with the transcriptional coactivator p300/CBP to enhance transcriptional activity. NF-κB dimer assembly with IκB, as well as its DNA binding and transcriptional activities, are regulated by p300/CBP acetyltransferases that principally target Lys218, Lys221 and Lys310 (12-14). This process is reciprocally regulated by histone deacetylases (HDACs); several HDAC inhibitors have been shown to activate NF-κB (12-14). T-cell co-stimulation and Calyculin A have both been shown to increase Ser468 phosphorylation (15,16). IKKβ (but not IKKα) and GSK-3β both target this site (16,17), which appears to have a negative regulatory role not involving inhibition of nuclear translocation after TNF-α or IL-1β stimulation (17). p65 phosphorylation at Ser536 regulates activation, nuclear localization, protein-protein interactions, transcriptional activity, and apoptosis (18-22).
- Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
- Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
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- Thompson, J.E. et al. (1995) Cell 80, 573-82.
- Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.
- Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
- Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.
- Chen, Z.J. et al. (1996) Cell 84, 853-62.
- Senftleben, U. et al. (2001) Science 293, 1495-9.
- Coope, H.J. et al. (2002) EMBO J 21, 5375-85.
- Xiao, G. et al. (2001) Mol Cell 7, 401-9.
- Ashburner, B.P. et al. (2001) Mol Cell Biol 21, 7065-77.
- Mayo, M.W. et al. (2003) J Biol Chem 278, 18980-9.
- Chen, L.F. et al. (2002) EMBO J 21, 6539-48.
- Mattioli, I. et al. (2004) Blood 104, 3302-4.
- Buss, H. et al. (2004) J Biol Chem 279, 49571-4.
- Schwabe, R.F. and Sakurai, H. (2005) FASEB J 19, 1758-60.
- Doyle, S.L. et al. (2005) J Biol Chem 280, 23496-501.
- Sasaki, C.Y. et al. (2005) J Biol Chem 280, 34538-47.
- Mattioli, I. et al. (2004) J Immunol 172, 6336-44.
- Bae, J.S. et al. (2003) Biochem Biophys Res Commun 305, 1094-8.
- Buss, H. et al. (2004) J Biol Chem 279, 55633-43.
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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Select rabbit monoclonal antibodies are developed, validated, and produced at CST using in part technology under license (granting certain rights including those under U.S. Patents No. 5,675,063 and in some instances 7,429,487) from Epitomics, Inc.