Yeow Karen, Novo-Perez Laurence, Gaillard Pascale, Page Patrick, Gotteland Jean-Pierre, Scheer Alexander, Lang Paul
Serono Pharmaceutical Research Institute, Plan-les-Ouates, Switzerland.
Assay Drug Dev Technol. 2006 Aug;4(4):451-60. doi: 10.1089/adt.2006.4.451.
Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that exists as two isozymes, GSK3alpha and GSK3beta. It plays important roles in regulating cell structure, function, and survival, and dysregulation of its function is linked to disorders such as Alzheimer's disease and type II diabetes. In resting cells, GSK3 is active and regulates the function of many downstream targets, including beta-catenin. We describe the development of a cell-based assay designed to measure the activity of GSK3 by directly measuring the accumulation of beta-catenin in Chinese hamster ovary clone K1 (CHOK1) cells. Beta-catenin levels were assessed using an antibody-based staining protocol with a luminometric readout. The assay is set up in a 96-well format. The use of GSK3 inhibitors demonstrated that this assay could be used to compare the effects of various small molecules on GSK3 inhibition in CHOK1 cells.
糖原合酶激酶-3（GSK3）是一种丝氨酸-苏氨酸蛋白激酶，以两种同工酶形式存在，即GSK3α和GSK3β。它在调节细胞结构、功能和存活方面发挥重要作用，其功能失调与阿尔茨海默病和II型糖尿病等疾病有关。在静息细胞中，GSK3具有活性，并调节许多下游靶点的功能，包括β-连环蛋白。我们描述了一种基于细胞的检测方法的开发，该方法旨在通过直接测量中国仓鼠卵巢克隆K1（CHOK1）细胞中β-连环蛋白的积累来测量GSK3的活性。使用基于抗体的染色方案并通过发光读数来评估β-连环蛋白水平。该检测以96孔板形式进行设置。GSK3抑制剂的使用表明，该检测可用于比较各种小分子对CHOK1细胞中GSK3抑制的影响。
