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Synthesis is included in this paper, and in a pending patent application publication.
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4. Each phosphocellulose filter was transferred into a separate scintillation vial, 1 mL cytoscint fluid was added, and the sample counted on a Packard 1900TR liquid scintillation analyzer for 1 min.
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2942618768
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The extracellular region and transmembrane domain of the human EGFR (amino acids 1-668) were combined with the intracellular region of RET (RET9 [amino acids 658-1072]). The human neuroblastoma cell line, SK-N-MC, was utilized to establish cell lines expressing the EGFR/RET construct and an empty vector control. The assay was performed by pre-incubating EGFR/RET-expressing cells for 30 min in serum-free MEM with increasing concentrations of GW569998X, GW585550A, or GW559768X and then activating RET by the addition of human epidermal growth factor (EGF) (100 ng/mL) for 10 min. Experimental incubations were terminated by collecting and directly lysing cells in 1× Laemmli (1% SDS, 10% glycerol, 100 mM dithiothreitol, and 50 mM Tris, pH 6.8) for Western blots. Western blots were sequentially probed for phospho-RET (Tyr-1062-R), RET9 (C-19) (Santa Cruz Biotechnology, Santa Cruz, CA), and actin (MAB1501) (Chemicon International, Temecula, CA). Yeatman, T. J. Nat. Rev. Cancer 2004, 4, 470-480.
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More indepth biological evaluation of these ret kinase inhibitors is included in a submitted manuscript by Michael A. Skinner, Jeffrey F. Moley, Karen E. Lackey, and Alex J. Freemerman, titled RET oncogene activation raises the apoptotic threshold in SK-N-MC cells.
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