For immunoblotting of total lysates, cells were first lysed in NP-40 buffer containing 150 mM NaCl, 50 mM tris (pH 7.6), and 1% (v/v) NP-40, supplemented with protease inhibitors (cOmplete, Roche) and phosphatase inhibitor cocktails. Lysates were boiled for 5 min after the addition of SDS sample buffer [60 mM tris-HCl (pH 6.8), 5% (v/v) glycerol, 1% (w/v) SDS, 2% (v/v) β-mercaptoethanol, and 0.02% (w/v) bromophenol blue] before SDS gel electrophoresis and immunoblotting. Primary antibodies against p-JAK2 (#3771), p-JAK1 (#3331), p-AKT S473 (#9271), p-ERK (#9101), p-STAT5 (#9359), Akt (#4685), and STAT5 (#9363) were purchased from Cell Signaling Technology. Primary antibodies against p-STAT1 (#612132), p-STAT3 (#612356), STAT3 (#612257), JAK1 (#610232), and phosphotyrosine-HRP conjugate (#610012) were obtained from BD Biosciences. A primary antibody against actin (#MAB1501) and anti-phosphotyrosine clone 4G10 (#05-777) were obtained from EMD Millipore. A primary antibody against TYK2 (#SC-169) and ubiquitin-HRP conjugate (SC-8017) were purchased from Santa Cruz Biotechnology. Primary antibodies against IL3Rα and βc were previously developed and characterized in our laboratory (25). Secondary antibodies were obtained from Thermo Fisher Scientific. The immunoprecipitation protocol has been described elsewhere (26). For immunoprecipitation, cells were lysed in NP-40 buffer containing 150 mM NaCl, 50 mM tris (pH 7.6), 1% (v/v) NP-40, and 50 mM iodoacetamide supplemented with protease inhibitors (cOmplete, Roche) and phosphatase inhibitor cocktails. For TUBE assays, cells were pretreated with MG132 (Sigma-Aldrich) to prevent degradation of ubiquitinated proteins for 10 min and stimulated with IL-3 in the presence or absence of ruxolitinib. Cells were lysed according to the manufacturer’s recommendations, and Agarose-TUBE (LifeSensors) was added for 2 hours. After two washes with lysis buffer, SDS sample buffer was used to elute bound ubiquitinated proteins, which were subsequently analyzed by immunoblotting with different JAK antibodies. Immunoblot analysis is representative of three independent experiments, with the exception of patient samples, where n = 4.

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