Abstract
A key regulatory node in NF-κB signaling is the removal of the IκBα inhibitor, whose levels are tightly controlled by the ubiquitin–proteasome system. In response to signal activation and transmission, ubiquitin E1, E2, and E3 enzymes are employed to generate a lysine 48-linked ubiquitin chain that triggers degradation of IκBα by the proteasome. In this chapter we describe an in vitro biochemical approach to reconstitute the ubiquitination system. To do so, we detail methods for the preparation of the relevant enzymes and substrate, as well as for the execution of the reaction with high efficiency. This sensitive and highly reproducible readout can be applied to the study of proteins, small molecules, and other factors that modulate IκBα ubiquitination, thereby producing outcomes that impact NF-κB signaling to advance the course of improving human health.
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Acknowledgements
We thank J. Nayak and other members of the Pan lab for their assistance with protocols and careful reading of the method. We are grateful to J. Hurwitz and I. Tappin for assistance with baculovirus preparation. R.A.C. was supported by NIH fellowship 1F30DK095572-01. Z.-Q.P. is the receipt of the 2013 Jiangsu special medical expert award. This work was supported by Public Health Service grants GM61051 and CA095634 to Z.-Q. P.
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Chong, R.A., Wu, K., Kovacev, J., Pan, ZQ. (2015). Generation of a Proteolytic Signal: E3/E2-Mediated Polyubiquitination of IκBα. In: May, M. (eds) NF-kappa B. Methods in Molecular Biology, vol 1280. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2422-6_20
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DOI: https://doi.org/10.1007/978-1-4939-2422-6_20
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