Jak2 activity is required for EpoR ubiquitination and targeting to the lysosomes. (A) Effect of Jak2 inhibition on EpoR ubiquitination. UT-7 cells were preincubated for 15 minutes with LLnL in the presence or absence of AG490 and were stimulated with Epo for the indicated times. Whole cell extracts were analyzed by Western blot using C-20 anti-EpoR antibodies. Number 1 indicates mature EpoR; 2, maturing EpoR; bracket, ubiquitinated EpoRs; arrowheads, nonspecific bands. (B) Effect of Jak2 inhibition on EpoR stability. UT-7 cells were preincubated with AG490 for 15 minutes and stimulated with Epo for the indicated times. Whole cell extracts were analyzed by Western blot using C-20 anti-EpoR antibodies. (C) Effect of Jak2 inhibition on Epo/EpoR trafficking. UT-7 cells were preincubated with AG490 (AG) alone (open symbols) or with AG490 and monensin (closed symbols) for 15 minutes before stimulation with 1 nM 125I-Epo. At the indicated times, cells were sampled for the determination of cell surface–associated radioactivity (squares, unbroken lines) and internalized radioactivity (triangles, dashed lines). Internalized radioactivity in control cells incubated without inhibitor is also indicated (open circles, dashed lines).