Analysis of nucleocytoplasmic shuttling of NF kappa B proteins in human leukocytes.
Controlled nucleocytoplasmic localization regulates activity of NF kappa B as well as other transcription factors. Analysis of the nucleocytoplasmic protein shuttling has been greatly facilitated by the use of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The authors have previously shown that LMB inhibits NF kappa B activity in human neutrophils by increasing the nuclear accumulation of NF kappa B inhibitor, I kappa B alpha. In this chapter, the authors describe a protocol that uses LMB to study the nucleocytoplasmic shuttling of I kappa B alpha in human macrophage-like U937 cells, thus inhibiting NF kappa B activity. This protocol should be readily adaptable to analyze the nucleocytoplasmic shuttling of other proteins in human leukocytes.
Ghosh
Chandra C
CC
Department of Biological Sciences, St. John's University, Queens, NY, USA.
Vu
Hai-Yen
HY
Mujo
Tomas
T
Vancurova
Ivana
I
D015153:Blotting, Western; D002457:Cell Extracts; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006801:Humans; D020672:I-kappa B Proteins / Q000037:antagonists & inhibitors; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008967:Molecular Biology / Q000379:methods*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D020298:U937 Cells
D002457:Cell Extracts; D005231:Fatty Acids, Unsaturated; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; C038753:leptomycin B