Analysis and quantitation of NF-kappaB nuclear translocation in tumor necrosis factor alpha (TNF-alpha) activated vascular endothelial cells.
Nuclear factor-kappa B (NF-kappaB) 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-kappaBs, which prevent entry into the nucleus. Following cellular stimulation, the I-kappaBs are rapidly degraded, activating NF-kappaB. The active form of NF-kappaB 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-kappaB is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-kappaB (p65 subunit, p65-NF-kappaB) 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-kappaB protein in the nucleus determined biochemically. The appearance of p65-NF-kappaB 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-kappaB can be reliably determined from IOD measurements of the immunofluorescent labeled protein.
Fuseler
John W
JW
Department of Cell, Developmental Biology and Anatomy, University of South Carolina School of Medicine, 6439 Garner's Ferry Road, Columbia, SC 29209, USA. fuseler@gw.med.sc.edu
Merrill
Dana M
DM
Rogers
Jennifer A
JA
Grisham
Matthew B
MB
Wolf
Robert E
RE
D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins
D016328:NF-kappa B; D021122:Protein Subunits; D014409:Tumor Necrosis Factor-alpha