Biochemical Assays for IC50 Determination

Recombinant JAK1, JAK2, JAK3 and TYK2 (Invitrogen) were used to develop activity assays in 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 1 mM EDTA, and 0.01% BRIJ 35. The amount of JAK protein was determined per aliquot, maintaining initial velocity and linearity over time. ATP concentration corresponded with the experimentally determined Km value, and substrate concentration (ULight-conjugated JAK1 [Tyr1023] peptide; PerkinElmer) was set according to the manufacturer’s instruction. After 90 minutes’ incubation at room temperature, the amount of phosphorylated substrate was measured with the addition of 2 nM europium–anti-phosphotyrosine Ab (PerkinElmer) and 10 mM EDTA in Lance detection buffer (PerkinElmer). Compound IC₅₀ values were determined by incubation with enzymes and ATP at room temperature for 90 minutes.