Williamson Adam, Jin Lingyan, Rape Michael
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA.
Methods Mol Biol. 2009;545:301-12. doi: 10.1007/978-1-60327-993-2_19.
Ubiquitination and protein degradation regulate cell cycle progression in all eukaryotes. During mitosis, ubiquitination by the Anaphase-Promoting Complex/Cyclosome (APC/C) triggers sister chromatid separation and mitotic exit. The APC/C is tightly regulated by phosphorylation, ubiquitination, association of activators or inhibitors, and competitive binding of substrates. Much of our understanding of the mechanism of APC/C-dependent ubiquitination has been obtained from studies using extracts of Xenopus laevis eggs or synchronized human tissue culture cells. Here, we describe protocols to prepare extracts of synchronized human cells, and discuss experiments to use extracts for the biochemical analysis of APC/C-dependent ubiquitination.
泛素化和蛋白质降解调节所有真核生物的细胞周期进程。在有丝分裂期间，后期促进复合物/细胞周期体（APC/C）介导的泛素化触发姐妹染色单体分离和有丝分裂退出。APC/C受到磷酸化、泛素化、激活剂或抑制剂的结合以及底物的竞争性结合的严格调控。我们对APC/C依赖性泛素化机制的许多理解来自于使用非洲爪蟾卵提取物或同步化的人类组织培养细胞进行的研究。在这里，我们描述了制备同步化人类细胞提取物的方案，并讨论了使用提取物进行APC/C依赖性泛素化生化分析的实验。
