Product Pathways - NF-kB Signaling
NF-κB Non-Canonical Pathway Antibody Sampler Kit #4888
|Kit Includes||Quantity||Applications||Reactivity||MW (kDa)||Isotype|
|Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697||40 µl||W IHC-P IHC-F||H M R Mk (B)||85 IKK-alpha 87 IKK-beta||Rabbit IgG|
|IKKα Antibody #2682||40 µl||W IP||H M R Mk (B)||85||Rabbit|
|Phospho-NF-κB2 p100 (Ser866/870) Antibody #4810||40 µl||W IP||H M (R) (B) (Dg)||110||Rabbit|
|NF-κB2 p100/p52 Antibody #4882||40 µl||W IP||H M R Mk||52 (mature). 120 (precursor).||Rabbit|
|NIK Antibody #4994||40 µl||W IP||H M (R) (Mk) (B) (Hr)||125||Rabbit|
|RelB (C1E4) Rabbit mAb #4922||40 µl||W IP||H M R Mk||70||Rabbit IgG|
|TRAF2 Antibody #4712||40 µl||W||H M R Mk||53||Rabbit|
|TRAF3 Antibody #4729||40 µl||W||H M R Mk||62||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
Reactivity Key: H=Human M=Mouse R=Rat Mk=Monkey B=Bovine Dg=Dog Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Specificity / Sensitivity
The Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb detects IKKα only when phosphorylated at Ser176/180 and IKKβ only when phosphorylated at Ser177/181. The Phospho-NF-κB2 p100 (Ser866/870) Antibody detects transfected levels of NF-κB2 p100 only when phosphorylated at Ser866/870. The TRAF2, TRAF3, IKKα, RelB, and p100/p52 antibodies detect endogenous levels of total protein of their respective targets. The NIK antibody detects transfected levels of NIK regardless of modification state.
Western blot analysis of extracts from NIH/3T3, HeLa and PC12 cells using IKKα Antibody #2682.
Western blot analysis of extracts from TNF-α (#2169, 20 ng/ml) and Calyculin A (#9902, 50 nM) treated HeLa and NIH/3T3 cells, using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697.
Western blot analysis of extracts from THP1 and Ramos cells using TRAF2 Antibody #4712.
Western blot analysis of extracts from Raji (human), PC12 (rat) and C2C12 (mouse) cell lines using TRAF3 Antibody #4729.
Western blot analysis of extracts from HeLa cells transfected with wild-type or mutant NF-κB2 p100 (SS866/870AA) and with or without NIK, using Phospho-NF-κB2 p100 (Ser866/870) Antibody #4810 (upper) and total NF-κB2 p100/p52 Antibody #4882 (lower). The p100 constructs were generously provided by Dr. Warner Greene of the Gladstone Institute of Virology and Immunology, Dr. Shao-Cong Sun of The Pennsylvania State University College of Medicine, and Dr. Gutian Xiao of the University of Pittsburgh Medical Center.
Western blot analysis of extacts from HeLa cells, untransfected or transfected with HA-NIK, using NIK Antibody #4994.
This kit contains reagents to examine the activation state and total protein levels of key components in the noncanonical NF-κB pathway: TRAF2, TRAF3, NIK, IKKα, p100, and RelB.
Source / Purification
Antibodies to phospho-IKKα/β (Ser176/180) and phospho-p100 (Ser866/870) are produced by immunizing rabbits with synthetic phosphopeptides corresponding to amino acids surrounding the indicated residues of human IKKα and NFκB2 p100, respectively. Antibodies to TRAF2, TRAF3, NIK, IKKα, RelB, and p100/p52 are produced by immunizing rabbits with synthetic peptides corresponding to amino acids at the carboxy terminus of TRAF2, in a central region of TRAF3, adjacent to Gly659 of human NIK, at the amino terminus of human IKKα, surrounding Ser424 of human RelB, and at the amino terminus of human p100/p52, respectively. Polyclonal antibodies are purified by Protein A and peptide affinity chromatography.
Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, RelB, c-Rel, 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. The p50 and p52 products form dimeric complexes with Rel proteins. While p50 associates with many of the NF-κB family members, p52 tends to form dimers primarily with RelB. A plethora of stimuli such TNFα and LPS induce the canonical NF-κB pathway, characterized by the activation of the classical IκB Kinase (IKK) complex (containing IKKα, IKKβ, IKKγ, and ELKS), which then phosphorylates inhibitory IκB molecules, targeting them for rapid degradation through a ubiquitin-proteasome pathway (3).The noncanonical pathway, triggered by BAFF, CD40L, and certain other stimuli, is based on the inducible phosphorylation and proteasome-mediated partial degradation of NF-κB2 p100 to p52, a process regulated by the NF-κB Inducing Kinase (NIK) and IKKα, but not IKKβ or IKKγ (4-6). NIK phosphorylates IKKα at Ser176/180 (6) and p100 at Ser866/870, then recruits IKKα to p100 where IKKα phosphorylates additional residues in the N- and C-terminus (8), leading to the ubiquitination and processing of p100 (9). The TNF Receptor Associated Factor molecules TRAF2 and TRAF3 have been shown to be negative regulators of the noncanonical pathway (10, 11), and their differential binding to receptors may also play a role in determining whether transduced signals activate the canonical pathway, noncanonical pathway, or both (12). TRAF3 promotes the rapid turnover of NIK in resting cells, and its activation-induced degradation is a key regulatory point in the pathway (13). This pathway is required for B cell maturation and activation, proper architecture of peripheral lymphoid tissue, and safeguards against autoimmunity (14).
- Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
- Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
- Ghosh, S. and Karin, M. (2002) Cell 109 Suppl, S81-96.
- Xiao, G. et al. (2001) Mol Cell 7, 401-9.
- Senftleben, U. et al. (2001) Science 293, 1495-9.
- Xiao, G. et al. (2001) EMBO J 20, 6805-15.
- Ling, L. et al. (1998) Proc Natl Acad Sci USA 95, 3792-7.
- Xiao, G. et al. (2004) J Biol Chem 279, 30099-105.
- Liang, C. et al. (2006) Cell Signal 18, 1309-17.
- Xia, Z.P. and Chen, Z.J. (2005) Sci STKE 2005, pe7.
- Liao, G. et al. (2004) J Biol Chem 279, 26243-50.
- Morrison, M.D. et al. (2005) J Biol Chem 280, 10018-24.
- Qing, G. et al. (2005) J Biol Chem 280, 40578-82.
- Xiao, G. et al. (2006) Cytokine Growth Factor Rev 17, 281-93.
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For Research Use Only. Not For Use In Diagnostic Procedures.