Cell Signaling Technology

Product Pathways - NF-kB Signaling

NF-κB p65 (C22B4) Rabbit mAb #4764

Applications Reactivity Sensitivity MW (kDa) Isotype
W IF-IC F H M R Mk B (Dg) Endogenous 65 Rabbit IgG

Applications Key:  W=Western Blotting  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  B=Bovine  Dg=Dog
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

NF-κB p65 (C22B4) Rabbit mAb detects endogenous levels of total NF-κB p65 protein. This antibody may also cross-react with the p50 subunit.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human NF-κB p65.

Western Blotting

Western Blotting

Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033 (upper) and NF-κB p65 (C22B4) Rabbit mAb (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa (human), PC12 (rat) and Neuro2A (mouse) cell lines using NF-κB p65 (C22B4) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® NF-κB p65 siRNA II (+), or SignalSilence® NF-κB p65 siRNA I #6261 (+), using NF-κB p65 (C22B4) Rabbit mAb #4764 and α-Tubulin (11H10) Rabbit mAb #2125. NF-κB p65 (C22B4) Rabbit mAb confirms silencing of NF-κB p65 and α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of NF-κB p65 siRNA.


Flow Cytometry

Flow Cytometry

Flow cytometric analysis of OVCAR8 cells using NF-κB p65 (C22B4) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or TNF-α-treated (#8902, 20ng/ml for 20 min, right), using NF-κB p65 (C22B4) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Background

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).

  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.

Application References

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

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