Abstract
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.
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Acknowledgments
Work presented here was supported by Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center for MSKCC, the William Randolph Hearst Fund in Experimental Therapeutics, the Lillian S Wells Foundation, and an NIH/NCI Cancer Center Support Grant 5 P30 CA008748–44. We thank Dr. Nancy Liu-Sullivan for her help during the write-up stages of the manuscript and with the earlier versions of the assay schematic.
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Antczak, C., Djaballah, H. (2016). A High-Content Assay to Screen for Modulators of EGFR Function. In: Zegzouti, H., Goueli, S. (eds) Kinase Screening and Profiling. Methods in Molecular Biology, vol 1360. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3073-9_8
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DOI: https://doi.org/10.1007/978-1-4939-3073-9_8
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3072-2
Online ISBN: 978-1-4939-3073-9
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