Preparation of synchronized human cell extracts to study ubiquitination and degradation.
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.
Williamson
Adam
A
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA.
Jin
Lingyan
L
Rape
Michael
M
D064173:Anaphase-Promoting Complex-Cyclosome; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002457:Cell Extracts; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D043743:Ubiquitin-Protein Ligase Complexes / Q000502:physiology*; D054875:Ubiquitination
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome