Cell Signaling Technology

Product Pathways - NF-kB Signaling

NF-κB Pathway Sampler Kit #9936

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
IKKα Antibody #2682 40 µl W IP H M R Mk (B) 85 Rabbit
IKKβ (2C8) Rabbit mAb #2370 40 µl W H M R Mk 87 Rabbit IgG
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
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
IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 40 µl W IP IHC-P IF-IC F H M R Mk B Pg GP 39 Mouse IgG1
Phospho-IκBα (Ser32) (14D4) Rabbit mAb #2859 40 µl W IP H M R Mk (C) (B) (Dg) (Pg) (GP) 40 Rabbit IgG
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
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Anti-mouse IgG, HRP-linked Antibody #7076 100 µl Horse

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  C=Chicken  B=Bovine  Dg=Dog  Pg=Pig  GP=Guinea Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Phospho-IKKα/β, phospho-NF-κB p65, and phospho-IκBα antibodies recognize endogenous levels IKKα/β, p65, and IκB-α, respectively, only when phosphorylated at the indicated residues. They do not cross-react with other family members at physiological levels. Total IKKα, IKKβ, p65 and IκBα antibodies recognize endogenous levels of their respective targets regardless of phosphorylation state and do not cross-react with other family members at physiological levels.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, NIH/3T3 and PC-12 cells using IIKKβ (2C8) Rabbit mAb #2370.

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3, HeLa and PC12 cells, using IKKα Antibody #2682.

Western Blotting

Western Blotting

Western blot analysis of extracts from TNF-alpha and Calyculin A treated HeLa and NIH/3T3 cells, using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697.


Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or treated with TNF-α (#2169, 20 ng/ml) for 5 minutes, using Phospho-IκB-α (Ser32) (14D4) Rabbit mAb #2859 (upper), or IκBα (44D4) Rabbit mAb #4812 (lower).

Western Blotting

Western Blotting

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 Blotting

Western Blotting

Western blot analysis of extracts from HeLa, NIH/3T3 and PC12 cells, using IκB-α (L35A5) Mouse mAb (Amino-terminal Antigen) #4814.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb #8242.

Description

The NF-κB Pathway Sampler Kit contains reagents to examine the activation state and total protein levels of key proteins in the NF-κB pathway: IKKα, IKKβ, NF-κB p65/RelA and IκBα. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per primary antibody.

Source / Purification

Antibodies to IKKα and NF- κB p65 are produced by immunizing rabbits with synthetic peptides corresponding to the amino terminus of human IKKα and NF-κBp65/RelA. IKKβ is produced by immunizing animals with a synthetic peptide corresponding to residues at the carboxy terminus of human IKKβ protein. The mouse monoclonal antibody is produced by immunizing animals with a GST-IκB-α fusion protein corresponding the amino-terminus of human IκBα. Antibodies to phospho-IKKα/β (Ser176/180), phospho-p65 (Ser536), and phospho-IκBα (Ser32) are produced by immunizing rabbits with (synthetic phosphopeptides corresponding to amino acids surrounding the indicated target residues of human IKKα, p65, and IκBα, respectively. Polyclonal antibodies are purified by Protein A and peptide affinity chromatography.

Background

The transcriptional nuclear factor κB (NF-κB)/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins. Activation occurs via phosphorylation of IκBα at Ser32 and Ser36, resulting in the ubiquitin-mediated proteasome-dependent degradation of IκBα and the release and nuclear translocation of active NF-κB dimers. The regulation of IκBβ and IκBε is similar to that of IκBα, however, the phosphorylation and degradation of these proteins occurs with much slower kinetics. Phosphorylation of IκBβ occurs at Ser/Thr19 and Ser23, while IκBε can be phosphorylated at Ser18 and Ser22. The key regulatory step in this pathway involves activation of a high molecular weight IkappaB kinase (IKK) complex, consisting of three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase. Activation of IKK depends on phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (176 and 180 in IKKα). NF-κB-inducing kinase (NIK), TANK-binding kinase 1 (TBK1), and its homolog IKKε (IKKi), phosphorylate and activate IKKα and IKKβ.

The NF-κB family of transcription factors is comprised of five proteins in mammals, p65/RelA, c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52). p105 and p100 are proteolytically processed to produce p50 and p52, respectively. The 50 kDa active form is produced through proteolytic processing following IKK-mediated phosphorylation of p105 at multiple sites (Ser922, 924, 928 and 933), while p100's processing to p52 is induced by phosphorylation of Ser864 and Ser868. The p50 and p52 products form dimeric complexes with Rel proteins, which are then able to bind DNA and regulate transcription. Phosphorylation of p65/RelA at Ser276 by PKA C and MSK1 enhances transcriptional activity. p65 phosphorylation at Ser536 regulates activation, nuclear localization, protein-protein interactions, and transcriptional activity. PMA-induced NF-κB transcriptional activity is dependent on the region of p65 containing the potential phosphorylation sites Ser457, Thr458, Thr464 and Ser468. Phosphorylation of Ser468 by GSK-3β inhibits basal p65 activity.

  1. Yamamoto, Y. and Gaynor, R.B. (2004) Trends Biochem. Sci. 29, 72-79.
  2. Ghosh, S. and Karin, M. (2002) Cell 109, S81-S96.
  3. Viatour, P. et al. (2005) Trends Biochem. Sci. 30, 43-52.

Application References

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Protocols

Companion Products

Selected rabbit monoclonal antibodies are produced under license (granting certain rights including those under U. S. Patent No. 5,675,063 and/or U.S.S.N. 11/476,277) from Epitomics, Inc.

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

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