Validation of a cell-based screen for insulin receptor modulators by quantification of insulin receptor phosphorylation.
Stimulation of a cell with insulin initiates a signal transduction cascade that results in cellular activities that include phosphorylation of the receptor itself. Measurement of the degree of phosphorylation can serve as a marker for receptor activation. Receptor phosphorylation has been measured using Western blot analysis, which is very low throughput and not easily quantifiable. The goal of this project was to develop a cell-based assay to measure receptor phosphorylation in high throughput. This report describes a cell-based assay for insulin receptor phosphorylation that is robust and amenable to high-volume screening in a microwell format.
Minor
Lisa K
LK
Johnson & Johnson Pharmaceutical Research and Development, L L C, Spring House, PA 19477, USA. lminor@prdus.jnj.com
Westover
Lori
L
Kong
Yan
Y
Patel
Darpana
D
Wildey
Mary Jo
MJ
Conway
Bruce R
BR
Demarest
Keith T
KT
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007202:Indicators and Reagents; D007328:Insulin / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction
D007202:Indicators and Reagents; D007328:Insulin; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin