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Analyzing the regulation and function of ATM
journal contributionposted on 2023-05-17, 13:03 authored by Lavin, MF, Scott, SP, Kozlov, S, Nuri GuvenNuri Guven
We describe here the cloning of full-length ataxia-telangiectasia mutated (ATM) cDNA and characterization of its activity. Full-length ATM cDNA is cloned into an inducible EBV-based vector (pMEP4) and its expression analyzed in a stably transfected cell line. ATM protein induction is monitored by immunoblotting with antibodies against both ATM and a FLAG sequence tag in the recombinant protein. Extracts from irradiated cells are immunoprecipitated with anti-ATM antibodies, and protein kinase activity is measured using p53(1-44)-specific substrate or by immunoblotting extracts with an anti-phosphoserine 15 p53-specific antibody. Missense mutations affecting ATM kinase activity are detected using in vitro mutagenesis of ATM cDNA followed by the procedures outlined above.
Publication titleMethods in Molecular Biology
Department/SchoolSchool of Pharmacy and Pharmacology
PublisherHumana Press, Inc.
Place of publicationUnited States
Rights statementCopyright 2004 Humanities Press Inc.