Interaction between cultured endothelial cells and macrophages: in vitro model for studying flavonoids in redox-dependent gene expression

Methods Enzymol. 2001:335:387-97. doi: 10.1016/s0076-6879(01)35261-8.

Abstract

This article focused on two methods to measure the activity of NF-kB. Both methods evalute "post-IkB phosphorylation" stages in the NF-kB activation cascade. In fact, EMSA performed with nuclear extracts provides an information only on NF-kB nuclear translocation and its ability to bind kB-DNA sequences. Likewise, the reporter gene assay is limited to assessing NF-kB-dependent gene expression no matter the mechanism that originally activated NF-kB. Nevertheless, the latter assay represents a more physiological and more reproducible way of measuring NF-kB activity in mammalian cells than the EMSA does. In order to obtain further insights into NF-kB signal transduction pathways, investigating IkB degradation and phosphorylation are recommended. The cloning and characterization of IkB kinases provided new testing possibilities based on measure of their activity.

MeSH terms

  • Animals
  • Cells, Cultured
  • Coculture Techniques
  • Endothelium / cytology*
  • Flavonoids / pharmacology*
  • Gene Expression Regulation / drug effects*
  • Humans
  • Macrophages / cytology*
  • Mice
  • NF-kappa B / metabolism
  • Oxidation-Reduction
  • Oxidative Stress
  • Reactive Oxygen Species
  • Transcriptional Activation / drug effects

Substances

  • Flavonoids
  • NF-kappa B
  • Reactive Oxygen Species