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GST-ITK and LCK-HIS full length enzymes were from Invitrogen (PV3875 and P3043, respectively) and substrate was BLK peptide (Ac-EFPIYDFLPAKKK-NH2). Reactions were carried out in a final volume of 51 μl with 50 mM HEPES buffer (pH 7.2), 15 mM MgCl2, 2 mM DTT, 0.015% Brij-35, 1 nM ITK or 0.5 nM LCK, 2 μM substrate, 20 μM ATP, and test article in a final concentration of 2% DMSO. After 35 min incubation at RT, reactions were stopped upon addition of 10 μl of 30% TCA. Samples were centrifuged (4350 rpm, 4 °C, 5 min) and subjected to LC/MS analysis on a Waters Acquity UPLC/TQD system equipped with a Waters Acquity UPLC BEH C18 (2.1 × 50 mm) 1.7 μm column (injection volume: 5 μl, column temperature: 60 °C, flow rate: 1 ml/min, solvent A: 0.1% formic acid in LC/MS grade water, solvent B: 0.1% formic acid in LC/MS grade ACN). Analytes were separated by applying a gradient from 15% to 32% solvent B within 0.7 min and detected in positive mode ESI-MS/MS by MRM (multiple reaction monitoring) of transitions 819.8/84.8 (BLK substrate) and 859.0/84.8 as well as 859.0/120.7 (phosphorylated BLK product). Ki values were determined using the Morrison tight-binding equation (J.W. William, and J.F. Morrison Methods Enzymol. 63 1979 437), modified to account for an ATP-competitive mechanism of inhibition and for the concentration of active kinase used
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