A cellular assay for measuring the inhibition of glycogen synthase kinase-3 via the accumulation of beta-catenin in Chinese hamster ovary clone K1 cells.
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.
Yeow
Karen
K
Serono Pharmaceutical Research Institute, Plan-les-Ouates, Switzerland. Karen.Yeow@phocus.com
Novo-Perez
Laurence
L
Gaillard
Pascale
P
Page
Patrick
P
Gotteland
Jean-Pierre
JP
Scheer
Alexander
A
Lang
Paul
P
D000627:Aminophenols / Q000494:pharmacology; D000818:Animals; D001552:Benzazepines / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D015153:Blotting, Western; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D020717:Eukaryotic Initiation Factor-2B / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology; D008163:Luminescent Measurements / Q000379:methods; D008301:Maleimides / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015203:Reproducibility of Results; D051176:beta Catenin / Q000737:chemistry / Q000378:metabolism*
C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; D000627:Aminophenols; D001552:Benzazepines; D001562:Benzimidazoles; C467064:CHIR98023; D004791:Enzyme Inhibitors; D020717:Eukaryotic Initiation Factor-2B; D007093:Imidazoles; D007211:Indoles; D008301:Maleimides; D011725:Pyridines; D011743:Pyrimidines; C417521:SB 216763; C120793:alsterpaullone; D051176:beta Catenin; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride; D004121:Dimethyl Sulfoxide