American Society of Hematology

Fig. 5.

$Fig. 5. Jak2 is involved in EPO-induced Stat1 and Stat3 activation. / (A) The effect of AG490 on EPO-induced activation of Jak2. UT-7/EPO cells were starved for 8 hours and subsequently treated with the indicated concentration of AG490. Then, 16 hours later, the cells were stimulated with EPO (10 U/mL) for 7 minutes, and the supernatants were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane. Next, the membrane was blotted with antiphosphoJak2 antibody. The same membrane was stripped and reblotted with anti-Jak2 antibody to confirm equal loading of protein. (B) The effect of AG490 on EPO-induced Stat1 and Stat3 activation. UT-7/EPO cells were starved for 8 hours and subsequently treated with the indicated concentration of AG490. Then, 16 hours later, the cells were stimulated with EPO (10 U/mL) for 15 minutes, and the nuclear fractions were extracted for EMSA. (C) The effect of AG490 on expression of Stat proteins. UT-7/EPO cells were starved for 8 hours and subsequently treated with the indicated concentration of AG490. Then, 16 hours later, the cells were stimulated with EPO (10 U/mL) for 15 minutes, and total cell lysates were prepared for Western blotting with each anti-Stat antibody.$

Jak2 is involved in EPO-induced Stat1 and Stat3 activation.

(A) The effect of AG490 on EPO-induced activation of Jak2. UT-7/EPO cells were starved for 8 hours and subsequently treated with the indicated concentration of AG490. Then, 16 hours later, the cells were stimulated with EPO (10 U/mL) for 7 minutes, and the supernatants were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane. Next, the membrane was blotted with antiphosphoJak2 antibody. The same membrane was stripped and reblotted with anti-Jak2 antibody to confirm equal loading of protein. (B) The effect of AG490 on EPO-induced Stat1 and Stat3 activation. UT-7/EPO cells were starved for 8 hours and subsequently treated with the indicated concentration of AG490. Then, 16 hours later, the cells were stimulated with EPO (10 U/mL) for 15 minutes, and the nuclear fractions were extracted for EMSA. (C) The effect of AG490 on expression of Stat proteins. UT-7/EPO cells were starved for 8 hours and subsequently treated with the indicated concentration of AG490. Then, 16 hours later, the cells were stimulated with EPO (10 U/mL) for 15 minutes, and total cell lysates were prepared for Western blotting with each anti-Stat antibody.

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