Human JAK2 complementary DNA (cDNA) containing a C-terminal Flag epitope tag was purchased from Sino Biological Inc., and the V617F and K882E kinase inactivation mutation were generated by PCR. JAK2-FLAG cDNA fragments were cloned into the pRufBlast retroviral expression vector to produce the pRufBlast:JAK2-Flag expression plasmids that were transfected into γ2A cells using Lipofectamine 2000 (Invitrogen) and then selected with (5 μg/ml) blasticidin to create stable cell lines.

Wild-type IL3Rα cDNA was cloned into the retroviral expression vector pRufHygro to produce pRufHygro:IL3Rα. Wild-type βc cDNA was cloned into the pRufPuro retroviral expression vector to produce pRufPuro:βc. The pRufHygro:IL3Rα and pRufPuro:βc plasmids were cotransfected into γ2A cells using Lipofectamine 2000 (Invitrogen) and then selected with hygromycin and puromycin to create stable cell lines. After selection, pools of hygromycin- and puromycin-resistant cells were sorted for IL3Rα and βc expression by flow cytometry. ON-TARGETplus Human JAK1 siRNA from Dharmacon was transfected using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen).

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