Figure 3
Figure 3. JAK2V617I induces only weak constitutive activation but marked cytokine hyperresponsiveness. (A) A representative analysis (1/2 independent experiments with similar results) of Ba/F3 EpoR cells that were engineered to overexpress equal levels of wild-type JAK2, JAK2V617F, or JAK2V617I by bicistronic retroviral transduction and cell sorting. Cells growing in medium supplemented with IL3 (left panel) or cells that acquired autonomous growth, namely cells expressing JAK2V617F or JAK2V617I (right panel, cells are denoted “Sel.”) were starved for 5 hours without serum and cytokines and then stimulated with EPO (20 U/mL) as indicated for 15 minutes, and then lysed in 1% NP40 buffer and assessed by Western blotting for specific phosphorylation at sites that reflect activation of JAK2, STAT1, STAT3, STAT5, and Erk1/2, and for total level of JAK2, STAT3, Erk1/2, HA, and β-actin protein expression. (B) A representative analysis (1/2 independent experiments with similar results) of Ba/F3 TpoR cells that were engineered to overexpress equal levels of wild-type JAK2, JAK2V617F, or JAK2V617I by bicistronic retroviral transduction and cell sorting. Cells growing in medium supplemented with IL3 were starved for 5 hours without serum and cytokines and then stimulated with Tpo (20 ng/mL) as indicated, and then lysed after 15 minutes in 1% NP40 buffer and assessed by Western blotting for specific phosphorylation at sites that reflect activation of JAK2, STAT1, STAT3, STAT5, and Erk1/2, and for total level of JAK2, STAT3, Erk1/2, HA, and β-actin protein expression. (C-D) Results of luciferase assays in γ2A fibrosarcoma JAK2-deficient cells transfected with cDNAs coding for TpoR (C) or EpoR (D) along with wild-type JAK2, JAK2V617F, or JAK2V617I, along with STAT5 and STAT-dependent firefly luciferase and pRLTK-driven renilla luciferase (rlu), for normalization. Dual luciferase was measured 24 hours after transfection. Results of 1 representative experiment of 3 independent experiments are shown. Data are expressed as means of triplicates, and error bars indicate SD. *P < .05, **P < .01, ***P < .001.

JAK2V617I induces only weak constitutive activation but marked cytokine hyperresponsiveness. (A) A representative analysis (1/2 independent experiments with similar results) of Ba/F3 EpoR cells that were engineered to overexpress equal levels of wild-type JAK2, JAK2V617F, or JAK2V617I by bicistronic retroviral transduction and cell sorting. Cells growing in medium supplemented with IL3 (left panel) or cells that acquired autonomous growth, namely cells expressing JAK2V617F or JAK2V617I (right panel, cells are denoted “Sel.”) were starved for 5 hours without serum and cytokines and then stimulated with EPO (20 U/mL) as indicated for 15 minutes, and then lysed in 1% NP40 buffer and assessed by Western blotting for specific phosphorylation at sites that reflect activation of JAK2, STAT1, STAT3, STAT5, and Erk1/2, and for total level of JAK2, STAT3, Erk1/2, HA, and β-actin protein expression. (B) A representative analysis (1/2 independent experiments with similar results) of Ba/F3 TpoR cells that were engineered to overexpress equal levels of wild-type JAK2, JAK2V617F, or JAK2V617I by bicistronic retroviral transduction and cell sorting. Cells growing in medium supplemented with IL3 were starved for 5 hours without serum and cytokines and then stimulated with Tpo (20 ng/mL) as indicated, and then lysed after 15 minutes in 1% NP40 buffer and assessed by Western blotting for specific phosphorylation at sites that reflect activation of JAK2, STAT1, STAT3, STAT5, and Erk1/2, and for total level of JAK2, STAT3, Erk1/2, HA, and β-actin protein expression. (C-D) Results of luciferase assays in γ2A fibrosarcoma JAK2-deficient cells transfected with cDNAs coding for TpoR (C) or EpoR (D) along with wild-type JAK2, JAK2V617F, or JAK2V617I, along with STAT5 and STAT-dependent firefly luciferase and pRLTK-driven renilla luciferase (rlu), for normalization. Dual luciferase was measured 24 hours after transfection. Results of 1 representative experiment of 3 independent experiments are shown. Data are expressed as means of triplicates, and error bars indicate SD. *P < .05, **P < .01, ***P < .001.

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