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
Part of the Methods in Molecular Biology book series (MIMB, volume 809)


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 



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


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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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