Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Real-time imaging of ligand-induced IKK activation in intact cells and in living mice


The transcription factor NF-κB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-κB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IκBα degradation by the 26S proteasome is a critical NF-κB regulatory control point, which is emerging as an important target for drug development. To directly monitor regulation of IKK activation in intact organisms, we engineered an IκBα–firefly luciferase (IκBα-FLuc) fusion reporter. In cultured cells and living animals, the reporter provided a continuous, noninvasive readout of the kinetics of ligand-induced IKK activation and the pharmacodynamics of selective inhibitors of both IKK and the 26S proteasome. This IκBα-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-κB transcriptional reporters for more complete temporal and regional investigations of the NF-κB signaling pathway in health and disease.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of stimulus-induced degradation of IκBα-FLuc.
Figure 2: Validation of IκBα-FLuc functionality in vitro.
Figure 3: Characterization of steady-state turnover of the IκBα-FLuc reporter.
Figure 4: Time- and concentration-dependent effects of various NF-κB modulators on TNFα-induced IKK activation and NF-kB transactivation.
Figure 5: Imaging pharmacological modulation of LPS-induced IKK activation in living mice.
Figure 6: Real-time imaging of IKK inhibition in tumors of PS-1145-treated mice.


  1. Karin, M., Cao, Y., Greten, F.R. & Li, Z.W. NF-κB in cancer: from innocent bystander to major culprit. Nat. Rev. Cancer 2, 301–310 (2002).

    Article  CAS  Google Scholar 

  2. Li, Q. & Verma, I.M. NF-κB regulation in the immune system. Nat. Rev. Immunol. 2, 725–734 (2002).

    Article  CAS  Google Scholar 

  3. Aggarwal, B.B. Nuclear factor κB: the enemy within. Cancer Cell 6, 203–208 (2004).

    Article  CAS  Google Scholar 

  4. Ghosh, S. & Karin, M. Missing pieces in the NF-κB puzzle. Cell 109 (Suppl.), S81–S96 (2002).

    Article  CAS  Google Scholar 

  5. Karin, M. & Ben-Neriah, Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu. Rev. Immunol. 18, 621–663 (2000).

    Article  CAS  Google Scholar 

  6. Hayden, M.S. & Ghosh, S. Signaling to NF-κB. Genes Dev. 18, 2195–2224 (2004).

    Article  CAS  Google Scholar 

  7. Greten, F.R. et al. IKKβ links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 118, 285–296 (2004).

    Article  CAS  Google Scholar 

  8. Lawrence, T., Bebien, M., Liu, G.Y., Nizet, V. & Karin, M. IKKα limits macrophage NF-κB activation and contributes to the resolution of inflammation. Nature 434, 1138–1143 (2005).

    Article  CAS  Google Scholar 

  9. Hay, R.T. Modifying NEMO. Nat. Cell Biol. 6, 89–91 (2004).

    Article  CAS  Google Scholar 

  10. Karin, M., Yamamoto, Y. & Wang, Q.M. The IKK NF-κB system: a treasure trove for drug development. Nat. Rev. Drug Discov. 3, 17–26 (2004).

    Article  CAS  Google Scholar 

  11. Luker, K.E. et al. Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl. Acad. Sci. USA 101, 12288–12293 (2004).

    Article  CAS  Google Scholar 

  12. Jacobs, M.D. & Harrison, S.C. Structure of an IκBα/NF-κB complex. Cell 95, 749–758 (1998).

    Article  CAS  Google Scholar 

  13. Barroga, C.F., Stevenson, J.K., Schwarz, E.M. & Verma, I.M. Constitutive phosphorylation of IκBα by casein kinase II. Proc. Natl. Acad. Sci. USA 92, 7637–7641 (1995).

    Article  CAS  Google Scholar 

  14. Place, R.F., Haspeslagh, D., Hubbard, A.K. & Giardina, C. Cytokine-induced stabilization of newly synthesized IκB-α. Biochem. Biophys. Res. Commun. 283, 813–820 (2001).

    Article  CAS  Google Scholar 

  15. Henkel, T. et al. Rapid proteolysis of IκB-α is necessary for activation of transcription factor NF-κB. Nature 365, 182–185 (1993).

    Article  CAS  Google Scholar 

  16. Lin, Z.P. et al. Prevention of brefeldin A–induced resistance to teniposide by the proteasome inhibitor MG-132: involvement of NF-κB activation in drug resistance. Cancer Res. 58, 3059–3065 (1998).

    CAS  PubMed  Google Scholar 

  17. Luker, G., Pica, C., Song, J., Luker, K. & Piwnica-Worms, D. Imaging 26S proteasome activity and inhibition in living mice. Nat. Med. 9, 969–973 (2003).

    Article  CAS  Google Scholar 

  18. Tan, C. & Waldmann, T.A. Proteasome inhibitor PS-341, a potential therapeutic agent for adult T-cell leukemia. Cancer Res. 62, 1083–1086 (2002).

    CAS  Google Scholar 

  19. An, J., Fisher, M. & Rettig, M.B. VHL expression in renal cell carcinoma sensitizes to bortezomib (PS-341) through an NF-κB–dependent mechanism. Oncogene 24, 1563–1570 (2005).

    Article  CAS  Google Scholar 

  20. May, M.J. et al. Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex. Science 289, 1550–1554 (2000).

    Article  CAS  Google Scholar 

  21. Pierce, J.W. et al. Novel inhibitors of cytokine-induced IκBα phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J. Biol. Chem. 272, 21096–21103 (1997).

    Article  CAS  Google Scholar 

  22. Hideshima, T. et al. NF-κB as a therapeutic target in multiple myeloma. J. Biol. Chem. 277, 16639–16647 (2002).

    Article  CAS  Google Scholar 

  23. Lin, Y., Yao, S., Veach, R., Torgenson, T. & Hawiger, J. Inhibition of nuclear translocation of transcription factor NF-κB by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem. 270, 14255–14258 (1995).

    Article  CAS  Google Scholar 

  24. Streetz, K.L. et al. Lack of gp130 expression in hepatocytes promotes liver injury. Gastroenterology 125, 532–543 (2003).

    Article  CAS  Google Scholar 

  25. Liu, F., Song, Y. & Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999).

    Article  CAS  Google Scholar 

  26. Bross, P.F. et al. Approval summary for bortezomib for injection in the treatment of multiple myeloma. Clin. Cancer Res. 10, 3954–3964 (2004).

    Article  CAS  Google Scholar 

  27. Ravi, R. & Bedi, A. NF-κB in cancer—a friend turned foe. Drug Resist. Updat. 7, 53–67 (2004).

    Article  CAS  Google Scholar 

  28. Castro, A.C. et al. Novel IKK inhibitors: beta-carbolines. Bioorg. Med. Chem. Lett. 13, 2419–2422 (2003).

    Article  CAS  Google Scholar 

  29. Imbert, V. et al. Tyrosine phosphorylation of IκB-α activates NF-κB without proteolytic degradation of IκB-α. Cell 86, 787–798 (1996).

    Article  CAS  Google Scholar 

  30. Carlsen, H., Moskaug, J.O., Fromm, S.H. & Blomhoff, R. In vivo imaging of NF-κB activity. J. Immunol. 168, 1441–1446 (2002).

    Article  CAS  Google Scholar 

  31. Magness, S.T. et al. In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-κB activation using a novel gene-targeted enhanced GFP reporter gene mouse. J. Immunol. 173, 1561–1570 (2004).

    Article  CAS  Google Scholar 

Download references


The authors thank S. Gammon for insightful discussions and help with statistical analyses. Funded by National Institutes of Health grant P50 CA94056.

Author information

Authors and Affiliations


Corresponding author

Correspondence to David Piwnica-Worms.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gross, S., Piwnica-Worms, D. Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat Methods 2, 607–614 (2005).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing