Figure 1
Figure 1. JAK2V617F requires an intact FERM and SH2 domain for constitutive ligand-independent activation. (A) Proliferation of parental Ba/F3 cells (Ø) and Ba/F3 cells expressing WT-JAK2 (JAK2) or JAK2 containing the V617F (V617F), R426K (JAK2 mSH2), L40A/Y41A (JAK2 mFERM) single mutations, or the JAK2V617F+R426K (V617F mSH2) and JAK2V617F+L40A/Y41A (V617F mFERM) double mutations as indicated. Cell growth in the absence of IL-3 was quantified by the relative optical density (OD) after 96 hours using an MTS-based assay (top panel). Absolute cell numbers over time were measured in the absence of IL-3 by trypan blue exclusion (bottom panel). The figures represent 1 of 3 independent experiments. Values are expressed as mean of triplicates ± SEM. (B) Parental Ba/F3 and Ba/F3 cells expressing WT and mutant JAK2 as indicated and described in panel A were serum-starved for 12 hours and stimulated with IL-3 or vehicle for 5 minutes. Lysates were subjected to Western blotting with the indicated antibodies (top panel). Unstimulated and IL-3–stimulated cell lines expressing JAK2 constructs described in panel A were immunoprecipitated from whole-cell lysates using an anti-Flag antibody and immunoblotted with the indicated antibodies (bottom panel). Note that the detection of JAK2V617F mFERM required longer exposures compared with the other JAK2 constructs. (C) Cell lysates were prepared from γ2A cells stably expressing mock vector (Ø), JAK2V617F alone (V617F), IL-3Rβ chain (IL-3Rβ) alone, IL-3Rβ chain together with JAK2V617F (V617F+IL-3Rβ), and IL-3R β chain plus α chain (IL-3Rβ+α) alone and together with JAK2V617F (V617F+IL-3Rβ+α). Lysates were analyzed by Western blotting with the indicated antibodies. (D) Cell lysates of γ2A cell lines stably expressing control vector (Ø), WT-JAK2 (JAK2) and JAK2R426K (JAK2 mSH2) along with IL-3Rα and β chains (IL-3R) were immunoblotted with the indicated antibodies (left panel). γ2A cell lines stably expressing IL-3R, control vector (Ø), WT-JAK2 (JAK2), and JAK2R426K (JAK2 mSH2) were serum-starved for 12 hours followed by stimulation with IL-3 or vehicle. Cells were lysed and analyzed by immunoblotting with the indicated antibodies (right panel).

JAK2V617F requires an intact FERM and SH2 domain for constitutive ligand-independent activation. (A) Proliferation of parental Ba/F3 cells (Ø) and Ba/F3 cells expressing WT-JAK2 (JAK2) or JAK2 containing the V617F (V617F), R426K (JAK2 mSH2), L40A/Y41A (JAK2 mFERM) single mutations, or the JAK2V617F+R426K (V617F mSH2) and JAK2V617F+L40A/Y41A (V617F mFERM) double mutations as indicated. Cell growth in the absence of IL-3 was quantified by the relative optical density (OD) after 96 hours using an MTS-based assay (top panel). Absolute cell numbers over time were measured in the absence of IL-3 by trypan blue exclusion (bottom panel). The figures represent 1 of 3 independent experiments. Values are expressed as mean of triplicates ± SEM. (B) Parental Ba/F3 and Ba/F3 cells expressing WT and mutant JAK2 as indicated and described in panel A were serum-starved for 12 hours and stimulated with IL-3 or vehicle for 5 minutes. Lysates were subjected to Western blotting with the indicated antibodies (top panel). Unstimulated and IL-3–stimulated cell lines expressing JAK2 constructs described in panel A were immunoprecipitated from whole-cell lysates using an anti-Flag antibody and immunoblotted with the indicated antibodies (bottom panel). Note that the detection of JAK2V617F mFERM required longer exposures compared with the other JAK2 constructs. (C) Cell lysates were prepared from γ2A cells stably expressing mock vector (Ø), JAK2V617F alone (V617F), IL-3Rβ chain (IL-3Rβ) alone, IL-3Rβ chain together with JAK2V617F (V617F+IL-3Rβ), and IL-3R β chain plus α chain (IL-3Rβ+α) alone and together with JAK2V617F (V617F+IL-3Rβ+α). Lysates were analyzed by Western blotting with the indicated antibodies. (D) Cell lysates of γ2A cell lines stably expressing control vector (Ø), WT-JAK2 (JAK2) and JAK2R426K (JAK2 mSH2) along with IL-3Rα and β chains (IL-3R) were immunoblotted with the indicated antibodies (left panel). γ2A cell lines stably expressing IL-3R, control vector (Ø), WT-JAK2 (JAK2), and JAK2R426K (JAK2 mSH2) were serum-starved for 12 hours followed by stimulation with IL-3 or vehicle. Cells were lysed and analyzed by immunoblotting with the indicated antibodies (right panel).

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