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Kinase Assays
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Kinase activities were assayed in Buffer A or C (unless otherwise stated), at 30°C, at a final ATP concentration of 15 μM. Blank values were subtracted and activities calculated as pmoles of phosphate incorporated during a 10 min. incubation. The activities are usually expressed in % of the maximal activity, i.e. in the absence of inhibitors. Controls were performed with appropriate dilutions of dimethylsulfoxide. In a few cases phosphorylation of the substrate was assessed by autoradiography after SDS-PAGE. GSK-3α/β was purified from porcine brain by affinity chromatography on immobilized axin [S1]. It was assayed, following a 1/100 dilution in 1 mg BSA/ml 10 mM DTT, with 5 μl 40 μM GS-1 peptide, a specific GSK-3 substrate, (YRRAAVPPSPSLSRHSSPHQSpEDEEE, synthesized by the Peptide Synthesis Unit, Institute of Biomolecular Sciences, University of Southampton, U.K.), in buffer A, in the presence of 15 μM [γ-32P] ATP (3,000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 30 min. incubation at 30°C, 25 μl aliquots of supernatant were spotted onto 2.5 x 3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 sec. later, the filters were washed five times (for at least 5 min. each time) in a solution of 10 ml phosphoric acid/liter of water. The wet filters were counted in the presence of 1 ml ACS (Amersham) scintillation fluid.
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References:
1. Meijer, L., Skaltsounis, A. L., Magiatis, P., Polychronopoulos, P., Knockaert, M., Leost, M., Ryan, X. P., Vonica, C. A., Brivanlou, A., Dajani, R., Crovace, C., Tarricone, C., Musacchio, A., Roe, S. M., Pearl, L. and Greengard, P. (2003) GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol. 10, 1255-12662.
2. Tseng, A. S., Engel, F. B. and Keating, M. T. (2006) The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes. Chem Biol. 13, 957-963
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