Recombinant JAK2 kinase domain (270 nM) was mixed with 80 nM recombinant PTP1B and no inhibitor, ruxolitinib, CHZ868, or itacitinib. The mixture was incubated at room temperature, and at various time points, 15 μl of reaction mix was added to 5 μl of SDS-PAGE reducing buffer [50 mM tris-HCl (pH 7.4), 200 mM β-mercaptoethanol, 10% (v/v) glycerol, 4% (w/v) SDS, and 0.2% (w/v) bromophenol blue]. Samples were blotted using primary antibody specific for the phosphorylated JAK activation loop (sc-10176, Santa Cruz Biotechnology) and an infrared fluorescent secondary antibody (925-32211, LI-COR) and imaged using an Odyssey infrared imager (LI-COR). Coomassie staining was used to monitor JAK1 levels due to unavailability of an appropriate antibody.

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