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Analysis and Quantitation of NF-κB Nuclear Translocation in Tumor Necrosis Factor Alpha (TNF-α) Activated Vascular Endothelial Cells

Published online by Cambridge University Press:  16 May 2006

John W. Fuseler ,
Dana M. Merrill ,
Jennifer A. Rogers ,
Matthew B. Grisham  and
Robert E. Wolf
John W. Fuseler
Affiliation:
Department of Cell, Developmental Biology and Anatomy, University of South Carolina School of Medicine, 6439 Garner's Ferry Road, Columbia, SC 29209, USA
Dana M. Merrill
Affiliation:
Department of Medicine, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130, USA
Jennifer A. Rogers
Affiliation:
Department of Medicine, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130, USA
Matthew B. Grisham
Affiliation:
Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130, USA
Robert E. Wolf
Affiliation:
Overton Brooks Veterans Administration Medical Center, 510 E. Stoner Ave., Shreveport, LA 71101, USA
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Abstract

Nuclear factor–kappa B (NF-κB) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-κBs, which prevent entry into the nucleus. Following cellular stimulation, the I-κBs are rapidly degraded, activating NF-κB. The active form of NF-κB rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-κB is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-κB (p65 subunit, p65-NF-κB) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-κB protein in the nucleus determined biochemically. The appearance of p65-NF-κB in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-κB can be reliably determined from IOD measurements of the immunofluorescent labeled protein.

Keywords

NF-κBnuclear translocationHUVECsimage analysisimmunofluorescenceintegrated optical density
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 12 , Issue 3 , June 2006 , pp. 269 - 276
Copyright
© 2006 Microscopy Society of America

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