A High-Content Assay to Screen for Modulators of EGFR Function
Cell-based assays have the potential and advantage to identify cell-permeable modulators of kinase function, and hence provide an alternative to the conventional enzymatic activity-driven discovery approaches that rely on purified recombinant kinase catalytic domains. Here, we describe a domain-based high-content biosensor approach to study endogenous EGFR activity whereby EGF-induced receptor activation, subsequent trafficking, and internalization are imaged and quantified using time-dependent granule formation in cells. This method can readily be used to search for EGFR modulators in both chemical and RNAi screening; with potential applicability to other receptor tyrosine kinases.
Key wordsRNAi siRNA Iressa Kinase Inhibitor High-content assay EGFR Cancer Drug discovery INCA2000 INCA3000 Microscopy HTS
- 6.Lanzerstorfer P, Borgmann D, Schutz G et al (2014) Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances. PLoS One 9(3):e92151Google Scholar
- 8.Sigma-Aldrich: EGFR biosensor cell line. http://www.sigmaaldrich.com/catalog/product/sigma/cll1097. Accessed 11 Mar 2014