Assaying NF-κB activation and signaling from TNF receptors.
Tumor necrosis factor (TNF)-mediated activation of the NF-κB family of transcription factors is mediated by two receptors, TNFR1 and TNFR2. These two receptors have unique roles in response to TNF and have been the focus of new therapeutic strategies in a variety of diseases with an immune or an inflammatory component. This chapter describes in vitro methods to functionally identify which TNF receptor is initiating NF-κB activation. This will include antibody-mediated receptor blockade and RNAi-mediated gene silencing targeting the individual receptors. The NF-κB activation methods presented include standard, accepted assays for monitoring the sequential activation steps through the NF-κB signal transduction cascade, including IκBα degradation, NF-κB nuclear translocation, and transcriptional activation of gene expression.
Department of Medicine and Rammelkamp Center for Education and Research, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, OH, 44109, USA.
Zhenzhen
Z
Wu
Leslie A
LA
Bruggeman
D057134:Antibodies, Neutralizing / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation
D057134:Antibodies, Neutralizing; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D034741:RNA, Small Interfering; D018124:Receptors, Tumor Necrosis Factor; D000072000:NF-KappaB Inhibitor alpha