title: Reconstitution Of β-catenin degradation in Xenopus egg extract.
abstract: Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways(1-3). Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus egg extract. One method is visually informative ([(35)S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
authors: Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center.
authors: Tony W
authors: TW
authors: Chen
authors: Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center.
authors: Matthew R
authors: MR
authors: Broadus
authors: Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center.
authors: Stacey S
authors: SS
authors: Huppert
authors: Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center; Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine; ethan.lee@vanderbilt.edu.
authors: Ethan
authors: E
authors: Lee
mesh_terms: D000818:Animals; D005260:Female; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism; D014982:Xenopus laevis; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D051176:beta Catenin