Cell Signaling Technology

Product Pathways - NF-kappaB Signaling

NF-κB Pathway Sampler Kit #9936

Kit Includes Quantity Applications Reactivity MW (kDa) Source
IKKα Antibody # 2682 40 microliters W IP H M R Mk (B) 85 Rabbit
IKKβ (L570) Antibody (IP Preferred) # 2678 40 microliters W IP H M R Mk Hm B 87 Rabbit
Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb # 2697 40 microliters W IHC-P IHC-F H M R Mk (B) 85 IKK-alpha 87 IKK-beta Rabbit
NF-κB p65 (C22B4) Rabbit mAb # 4764 40 microliters W IHC-P IF-IC F H M R Mk B (Dg) 65 Rabbit
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb # 3033 40 microliters W IP IF-IC F H M R Mk (Dg) 65 Rabbit
IκB-α (L35A5) Mouse mAb (Amino-terminal Antigen) # 4814 40 microliters W IP IHC-P IF-IC F H M R Mk B 39 Mouse
Phospho-IκBα (Ser32) (14D4) Rabbit mAb # 2859 40 microliters W IP H M R Mk 40 Rabbit
Anti-rabbit IgG, HRP-linked Antibody # 7074 100 microliters Goat
Anti-mouse IgG, HRP-linked Antibody # 7076 50 microliters Horse

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Hm=Hamster  B=Bovine  Dg=Dog

Specificity / Sensitivity

Phospho-IKKα/β, phospho-NF-κB p65, and phospho-IκB-α 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 IκB-α 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 NIH/3T3 cells treated with 20 ng/ml TNFα #2169 for the indicated times, using IKKα Antibody #2682 and IKKβ (L570) Antibody (IP Preferred) #2678 (top left), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697 (top right), NF-κB p65 (C22B4) Rabbit mAb #4764 (center left), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033 (center right), IκB-α (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (lower left) and Phospho-IκB-α (Ser32) (14D4) Rabbit mAb #2859 (lower right).

Source / Purification

Antibodies to IKKα, IKKβ, and NF-κB p65 are produced by immunizing rabbits with KLH-coupled synthetic peptides corresponding to amino acids at the amino terminus of human IKKα, surrounding Leu570 of human IKKβ, and near the amino terminus of human p65, respectively. The mouse monoclonal antibody is produced by immunizing mice with a GST-IκB-α fusion protein corresponding the amino-terminus of human IκB-α. Antibodies to phospho-IKKα/β (Ser176/180), phospho-p65 (Ser536), and phospho-IκB-α (Ser32) are produced by immunizing rabbits with KLH-coupled synthetic phospho-peptides corresponding to amino acids surrounding the indicated target residues of human IKKα, p65, and IκB-α, 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 IκB-α at Ser32 and Ser36, resulting in the ubiquitin-mediated proteasome-dependent degradation of IκB-α and the release and nuclear translocation of active NF-κB dimers. The regulation of IκB-β and IκB-ε is similar to that of IκB-α, however, the phosphorylation and degradation of these proteins occurs with much slower kinetics. Phosphorylation of IκB-β occurs at Ser/Thr19 and Ser23, while IκB-ε 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.

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