XB-ART-3442
Methods Mol Biol
2004 Jan 01;280:229-43. doi: 10.1385/1-59259-788-2:229.
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A Xenopus cell-free system for analysis of the Chfr ubiquitin ligase involved in control of mitotic entry.
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The checkpoint protein Chfr delays entry into mitosis in the presence of mitotic stress. We have analyzed the Chfr checkpoint pathway in the Xenopus cell-free system. We showed that Chfr is a ubiquitin ligase that targets polo-like kinase (Plk1) for degradation, leading to delayed activation of the Cdc25C phosphatase and prolonged inhibitory phosphorylation of Cdc2 at the G2/M transition. In this chapter, we will describe biochemical methods we developed to analyze the Chfr auto-ubiquitination activity and the ubiquitination of its substrate Plk1, as well as functional assays to investigate the Chfr pathway in Xenopus extracts.
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Species referenced: Xenopus
Genes referenced: cdc25c cdk1 chfr plk1