Abstract
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 p531–44-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.
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Lavin, M.F., Scott, S.P., Kozlov, S., Gueven, N. (2004). Analyzing the Regulation and Function of ATM. In: Schönthal, A.H. (eds) Checkpoint Controls and Cancer. Methods in Molecular Biology, vol 281. Humana Press. https://doi.org/10.1385/1-59259-811-0:163
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DOI: https://doi.org/10.1385/1-59259-811-0:163
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