Electrophoretic mobility shift assay analysis of NFκB transcriptional regulation by nuclear IκBα.

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.

• Authors: Ashish Juvekar, Sitharam Ramaswami, Subrata Manna, Tzu-Pei Chang, Adeel Zubair, Ivana Vancurova
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 809 Pg. 49-62 ( 2012) ISSN: 1940-6029 [Electronic] United States
• PMID: 22113267 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• I-kappa B Proteins
• NF-kappa B
• NFKBIA protein, human
• NF-KappaB Inhibitor alpha
• Proteasome Endopeptidase Complex
• ATP dependent 26S protease

Topics

• Blotting, Western
• Cell Line
• Electrophoresis, Polyacrylamide Gel
• Electrophoretic Mobility Shift Assay (methods)
• Humans
• I-kappa B Proteins (genetics, metabolism)
• NF-KappaB Inhibitor alpha
• NF-kappa B (genetics, metabolism)
• Proteasome Endopeptidase Complex (metabolism)