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Electrophoretic Mobility Shift Assay Analysis of NFκB Transcriptional Regulation by Nuclear IκBα

  • Ashish Juvekar
  • Sitharam Ramaswami
  • Subrata Manna
  • Tzu-Pei Chang
  • Adeel Zubair
  • Ivana VancurovaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 809)

Abstract

Transcription factor NFκB is a key regulator of genes involved in immune and inflammatory responses, as well as genes regulating cell proliferation and survival. In addition to many inflammatory disorders, NFκB is constitutively activated in a variety of human cancers and leukemia. Thus, inhibition of NFκB DNA binding activity represents an important therapeutic approach for disorders characterized by high levels of constitutive NFκB activity. We have previously shown that NFκB DNA binding activity is suppressed by the nuclear translocation and accumulation of IκBα, which is induced by inhibition of the 26S proteasome. In this chapter, we describe a protocol that uses small inhibitory RNA (si RNA) interference followed by electrophoretic mobility shift assay (EMSA) to analyze the regulation of NFκB DNA binding by nuclear IκBα induced by the proteasome inhibitor MG132. Using this protocol, we show that in human leukemia Hut-78 cells that exhibit high levels of NFκB DNA binding activity, MG132 induces nuclear translocation and accumulation of IκBα, which then specifically inhibits NFκB DNA binding. This protocol uses human leukemia Hut-78 cells; however, it can be easily adapted for other cells exhibiting high levels of constitutive NFκB DNA binding.

Key words

Electrophoretic mobility shift assay MG132 NFκB Nuclear IκBα Nuclear translocation Proteasome inhibition si RNA Transcriptional regulation 

Notes

Acknowledgements

This work was supported by NIH grants GM079581 and AI085497 to I.V.

References

  1. 1.
    Baeuerle, P, A. and Baltimore, D. (1996) NFκB: ten years after. Cell 87, 13–20.Google Scholar
  2. 2.
    Ghosh, S. and Karin, M. (2002) Missing pieces in the NFκB puzzle. Cell 109, S81–96.Google Scholar
  3. 3.
    Barnes, P. J. and Karin, M. (1997) NFκB – a pivotal transcription factor in chronic inflammatory diseases. New Engl. J. Med. 336, 10661071.Google Scholar
  4. 4.
    Baldwin, A. S. (2001) The transcription factor NFκB and human disease. J. Clin. Invest. 107, 3–6.Google Scholar
  5. 5.
    Yamamoto, Y. and Gaynor, R. B. (2001) Therapeutic potential of inhibition of the NFκB pathway in the treatment of inflammation and cancer. J. Clin Invest. 107, 135142.Google Scholar
  6. 6.
    Hayden, M. S. and Ghosh, S. (2008) Shared principles in NFκB signaling. Cell 132, 344–362.Google Scholar
  7. 7.
    Prasad, S., Ravindran, J. and Aggarwal, B. B. (2010) NFκB and cancer: how intimate is this relationship. Mol. Cell. Biochem. 336, 25–37.Google Scholar
  8. 8.
    Bharti, A. C. and Aggarwal, B. B. (2002) NFκB and cancer: its role in prevention and therapy. Biochem. Pharmacol. 64, 883–888.Google Scholar
  9. 9.
    Dutta, J., Fan, Y., Gupta, N., Fan, G. and Gelinas, C. (2006) Current insights into the regulation of programmed cell death by NFκB. Oncogene 25, 6800–6816.Google Scholar
  10. 10.
    Karin, M. (2006) NFκB in cancer development and progression. Nature 441, 431–436.Google Scholar
  11. 11.
    Wan, F. and Lenardo, M. J. (2010) The nuclear signaling of NFκB: current knowledge, new insights, and future perspectives. Cell. Res. 20, 24–33.Google Scholar
  12. 12.
    Lenz, G. and Staudt, L. M. (2010) Aggressive lymphomas. N. Engl. J. Med. 362, 1417–1429.Google Scholar
  13. 13.
    Mancino, A. and Lawrence, T. (2010) NFκB and tumor-associated macrophages. Clin. Cancer Res. 16, 784–789.Google Scholar
  14. 14.
    Karin, M. and Neriah, Y. B. (2000) Phosphorylation meets ubiquitination: The control of NF-κB activity. Annu. Rev. Immunol. 18, 621–663.Google Scholar
  15. 15.
    McConkey, D.J. and Zhu, K. (2008) Mechanisms of proteasome inhibitor action and resistance in cancer. Drug Resist. Updat. 11, 164–179.Google Scholar
  16. 16.
    Shah, J. J. and Orlowski, R. Z. (2009) Proteasome inhibitors in the treatment of multiple myeloma. Leukemia 23, 1964–1979.Google Scholar
  17. 17.
    Izban, K. F., Ergin, M., Qin, J. Z., Martinez, R. L., Pooley, R. J., Saeed, S. and Alkan, S. (2000) Constitutive expression of NFκB is a characteristic feature of mycosis fungoides: implications for apoptosis resistance and pathogenesis. Hum. Pathol. 31, 1482–1490.Google Scholar
  18. 18.
    Sors, A., Jean-Louis, F., Pellet, C., Laroche, L., Dubertret, L., Courtois, G., Bachelez, H. and Michel, L. (2006) Down-regulating constitutive activation of the NFκB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis. Blood 107, 2354–2363.Google Scholar
  19. 19.
    Sors, A., Jean-Louis, F., Bégué, E., Parmentier, L., Dubertret, L., Dreano, M., Courtois, G., Bachelez, H. and Michel, L. (2008) Inhibition of IκB kinase subunit 2 in cutaneous T-cell lymphoma down-regulates NFκB constitutive activation, induces cell death, and potentiates the apoptotic response to antineoplastic chemotherapeutic agents. Clin. Cancer Res. 14, 901–911.Google Scholar
  20. 20.
    Kiessling, M. K., Klemke, C. D., Kaminski, M. M., Galani, I. E., Krammer, P. H. and Gulow, K. (2009) Inhibition of constitutively activated NFκB induces reactive oxygen species and iron-dependent cell death in cutaneous T-cell lymphoma. Cancer Res. 69, 2365–2374.Google Scholar
  21. 21.
    Vu, H.Y., Juvekar, A., Ghosh, C., Ramaswami S., Le, D. H. and Vancurova, I. (2008) Proteasome inhibitors induce apoptosis of prostate cancer cells by inducing nuclear translocation of ΙκΒα. Arch. Biochem. Biophys. 475, 156–163.Google Scholar
  22. 22.
    Castro-Alcaraz, S., Miskolci, V., Kalasapudi, B., Davidson, D. and Vancurova, I. (2002) NFκB regulation in human neutrophils by nuclear IκBα: correlation to apoptosis. J. Immunol. 169, 3947–3953.Google Scholar
  23. 23.
    Ghosh, C. C., Ramaswami, S., Juvekar, A., Vu, H. Y., Galdieri, L., Davidson, D. and Vancurova, I. (2010) Gene specific repression of proinflammatory cytokines in stimulated human macrophages by nuclear IκBα. J. Immunol. 185, 3685–3693.Google Scholar
  24. 24.
    Vancurova, I., Miskolci, V. and Davidson, D. (2001) NFκB activation in TNFα-stimulated neutrophils is mediated by protein kinase Cδ. Correlation to nuclear IκBα. J. Biol. Chem. 276, 19746–19752.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ashish Juvekar
    • 1
  • Sitharam Ramaswami
    • 1
  • Subrata Manna
    • 1
  • Tzu-Pei Chang
    • 1
  • Adeel Zubair
    • 1
  • Ivana Vancurova
    • 1
    Email author
  1. 1.Department of Biological SciencesSt. John’s UniversityQueensUSA

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