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72049107896
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JAK2 Ki Determination: ATP and polyE4Y were obtained from Sigma Chemical Co, St. Louis, MO, USA, 33P-γ-ATP, GF/B filter plates, and Ultima Gold™ scintillant were purchased from Perkin-Elmer Life Sciences (Boston, MA, USA, JAK2 used in Vertex assays were expressed and purified by the Gene Expression and Protein Biochemistry groups, respectively, at Vertex Pharmaceuticals Incorporated using standard recombinant methods.a Methods: The inhibitory activity of compounds in Table 1 against JAK2 was determined by following the residual kinase activity of JAK2 using a radiometric assay. The final concentration of the components in the assay were as follows: 100 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% BSA, 0.6 nM JAK2, 0.5 mg/ml polyE4Y, and 12 μM 33P-γ-ATP. A stock solution of inhibitor was made up in DMSO from which additional dilutions were made in DMSO; a 1.5 μL aliquot of DMSO or inhibitor in DMSO was
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2 using Prism software, version 4.0, San Diego, CA, USA. (a) Fox, T.; Coll, J. T.; Ford, P. J.; Germann, U. A.; Porter, M. D.; Pazhanisamy, S.; Fleming, M. A.; Galullo, V.; Su, M.-S.; Wilson, K. P. Protein Sci. 1998, 7, 2249; (b) Morrison, J. F.; Stone, S. R. Comments Mol. Cell Biophys. 1985, 2, 347.
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72049128700
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note
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50 values were calculated with Softmax Pro (Molecular Devices, Sunnyvale CA).
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