Chapter title |
Measurement of NF-κB Activation in TLR-Activated Macrophages
|
---|---|
Chapter number | 5 |
Book title |
Innate Immune Activation
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7519-8_5 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7518-1, 978-1-4939-7519-8
|
Authors |
Orna Ernst, Sharat J. Vayttaden, Iain D. C. Fraser |
Abstract |
Nuclear factor kappa-B (NF-κB) is a key transcription factor in the regulation of the innate immune inflammatory response in activated macrophages. NF-κB functions as a homo- or hetero-dimer derived from one or more of the five members of the NF-κB family, and is activated through a well-studied process of stimulus-dependent inhibitor degradation, post-translational modification, nuclear translocation, and chromatin binding. Its activity is subject to multiple levels of feedback control through both inhibitor protein activity and direct regulation of NF-κB components. Many methods have been developed to measure and quantify NF-κB activation. In this chapter, we summarize available methods and present a protocol for image-based measurement of NF-κB activation in macrophages activated with microbial stimuli. Using either a stably expressed GFP-tagged fusion of the RelA NF-κB protein, or direct detection of endogenous RelA by immunocytochemistry, we describe data collection and analysis to quantify NF-κB cytosol to nuclear translocation in single cells using fluorescence microscopy. |
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