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.