Analysis and Quantitation of NF-[kappa]B Nuclear Translocation in Tumor Necrosis Factor Alpha (TNF-[alpha]) Activated Vascular Endothelial Cells

• Published in: Microscopy and microanalysis Vol. 12; no. 3; pp. 269 - 276
• Main Authors: Fuseler, John W., Merrill, Dana M., Rogers, Jennifer A., Grisham, Matthew B., Wolf, Robert E.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.06.2006
• Subjects: Binding sites; Charitable foundations; Fluorescence; Kinases; Proteins; Software; Translocation
• ISSN: 1431-9276; 1435-8115
• DOI: 10.1017/S1431927606060260

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. [PUBLICATION ABSTRACT]