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Kinase assays were typically performed at 25 °C by preincubating a 0.5 µM concentration of protein in 1 mM MnCl2 for 5 min in buffer (20 mM HEPES at pH 7.4, 50 mM NaCl) and then initiated by addition of 100 µM ATP. The reactions were stopped after the noted period (30 min) then did immunoprecipitation with pS237A (custom antibody Thermo Scientific). Proteins were resolved by SDS-PAGE and immunoblot with Total S237A (custom antibody Thermo Scientific), with enhanced chemiluminescence (ECL Thermo Scientific)35.
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