JAK domain (3 nM) was mixed with 1 mM STAT5b substrate peptide, 0.5 mM ATP, 1 μCi [γ-32P] ATP, 30 mM tris-HCl (pH 8.0), 100 mM NaCl, bovine serum albumin (0.2 mg/ml), 1 mM MgCl2, 1 mM tris(2-carboxyethyl)phosphine (TCEP), and various concentrations of JAK inhibitor. After incubation at room temperature for 20 to 60 min, 3.5 μl of reaction mix was spotted onto P81 phosphocellulose paper (Whatman, GE Healthcare), which was washed four times for 15 min with 100 ml of 5% (v/v) H3PO4. The paper was dried and exposed to a phosphorimager plate overnight, which was then scanned using a Typhoon FLA 7000 PhosphorImager (GE Life Sciences).

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