Methods Enzymol 2005 Jan 01;399:567-85. doi: 10.1016/S0076-6879(05)99038-1.

Identifying small molecule inhibitors of the ubiquitin-proteasome pathway in Xenopus egg extracts.

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Small molecule inhibitors of the proteasome have been crucial for dissecting the mechanism of proteasome-dependent protein degradation and identifying substrates of the ubiquitin-proteasome system (UPS). To identify small molecules that block ubiquitin-dependent protein degradation through other mechanisms, we have developed pathway-based screening approaches in Xenopus egg extracts. The regulated degradation of UPS substrates can be reconstituted in these extracts, providing an excellent system in which to perform forward chemical genetic screens. The ability to manipulate extracts biochemically and to compare the activity of small molecules across different assays facilitates the identification of potential target proteins. Here we describe methods for identifying inhibitors of the proteolytic pathways that regulate cell cycle progression and Wnt signaling in Xenopus extracts.

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