Detection of IκB degradation dynamics and IκB-α ubiquitination

The NF-κB signaling pathway is a primary regulator of infl ammation that has been implicated in the pathogenesis of immune disorders and cancer. This signaling network is strictly regulated; in a nonactivated state, NF-κB transcription factors are sequestered in the cytoplasm by the IκB family of proteins. Various pro-infl ammatory stimuli result in the phosphorylation and subsequent ubiquitination of IκBs. These events lead to rapid degradation of IκB and allow translocation of the transcription factors to the nucleus. Therefore, ubiquitination and degradation of IκBs are critical steps in NF-κB pathway activation and can serve as a quantitative parameter to assess pathway activation. In this article, we present a detailed protocol for the quantifi cation of in vivo ubiquitination and turnover of IκB-α in response to a variety of cellular stimuli.