Cell Signaling Technology

Product Pathways - NF-kappaB Signaling

SignalSilence® NF-κB p65 siRNA Kit (Human Specific) #6260

Kit Includes

SignalSilence NF-kappaB p65 siRNA (Human Specific) #6261
pre-validated single sequence siRNA duplex guaranteed to specifically inhibit NF-kappaB expression in specified cell lines.
SignalSilence® Control siRNA (Fluorescein Conjugate) #6201
a non-targeted negative control duplex designed to monitor transfection efficiency using microscopy and to monitor siRNA specificity.
Targeted NF-kappaB p65 Antibody #3034
to confirm silencing of NF-kappaB p65 expression.
Non-targeted p42 MAPK Antibody #9108
to control for protein loading and NF-kappaB p65 siRNA specificity.
*Transfection Reagent
designed specifically for highly efficient siRNA delivery in mammalian cells. Transfection reagent is supplied by Mirus.

*Store at 4°C (packaged separately)

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells transfected with nontargeted (-) or targeted (+) siRNA. p65 was detected using NF-kappaB p65 Antibody #3034, and p42 was detected using p42 MAPK Antibody #9108. The NF-kappaB p65 Antibody confirms silencing of p65 expression, and the p42 MAPK Antibody is used to control for loading and siRNA specificity.

Fluorescent Detection

Fluorescent Detection

Fluorescent detection of SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 in living HeLa cells 24 hours post-transfection, demonstrating nearly 100% transfection efficiency.

Description

SignalSilence® NF-kappaB p65 siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit NF-kappaB p65 expression. The kit utilizes RNA interference, a method in which gene expression can be selectivelysilenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA kits are rigorously tested in-house and have been shown to reduce protein expression. SignalSilence® NF-kappaB p65 siRNA Kit includes NF-kappaB p65 siRNA, a target-specific NF-kappaB p65 Antibody to confirm the silencing of NF-kappaB p65 expression and a non-target antibody (p42 MAPK) to control for loading and monitor specificity of NF-kappaB p65 siRNA. Additionally, a fluorescein-labeled non-targeted siRNA control allows the user to monitor transfection efficiency and siRNA specificity. The kit also contains a transfection reagent designed solely for efficient siRNA delivery in mammalian cells.

Directions for Use

CST recommends transfection with 100 nM human-specific NF-kappaB p65 siRNA 48 hours prior to cell lysis. See Protocol for transfection procedure.

Tested Cell Lines

HeLa

Background

Transcription factors of the nuclear factor kappa 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 an 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 is then translocated to the nucleus (9-11).

Small Interfering RNA (siRNA) has been used to specifically silence NF-kappaB p65 in colon cancer cells (10).

  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.
  12. Verma, U. N. et al. (2003) Clin. Cancer Res. 9, 1291-1300.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

Limited Use Label License, RNA interference: This product is licensed under European Patent 1144623 and foreign equivalents from Ribopharma AG, Kulmbach, Germany and is provided only for use in non-commercial research specifically excluding use (a) in drug discovery or drug development, including target identification or target validation, by or on behalf of a commercial entity, (b) for contract research or commercial screening services, (c) for the production or manufacture of siRNA-related products for sale, or (d) for the generation of commercial databases for sale to Third Parties. Information about licenses for these and other commercial uses is available from Ribopharma AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany.

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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