The SH2 domain is required for the transphosphorylation of JAK2V617F. (A) 2 WT-JAK2 or JAK2V617F constructs containing either a myc (M-JAK2, M-V617F) or Flag tag (F-JAK2, F-V617F) were coexpressed in 293T cells. Flag-tagged JAK2 proteins were immunoprecipitated from cell lysates and analyzed for co-IP of myc-tagged JAK2 by anti-myc immunoblotting and phospho-JAK2. (B) 293T cells coexpressing either Flag- and myc-tagged JAK2V617F (F-V617F, M-V617F) or Flag- and myc-tagged JAK2V617F+R426K (F-V617F mSH2, M-V617F mSH2) were analyzed as described in panel A. (C) Ba/F3 cells stably expressing Flag- and myc-tagged JAK2V617F and JAK2V617F+R426K mutants as indicated were lysed analyzed by Flag-IP and subsequent phospho-JAK2 and flag immunoblot. Expression levels of the JAK2 constructs in the lysates were controlled by Flag and myc Western blot. The cell lines used in this experiment were mock-transduced Ba/F3 cells (Ø, lane 1), flag-JAK2V617F (F-V617F) with myc-JAK2V617F (M-V617F; lane 2), flag-JAK2V617F+R426K (F-V617F mSH2) with myc-JAK2V617F+R426K (M-V617F mSH2; lane 3), and Flag-JAK2V617F+R426K (F-V617F mSH2) with myc-JAK2V617F (M-V617F; lane 4).