Induction of Stat-5 phosphorylation by GM-CSF or IL-3 in bcr-abl⁻ but not bcr-abl⁺ Mo7e cells.

Exogenous cytokines cannot reactivate Stat-5 in STI-571–treated MBA or K562 cells. (A) Mo7e or MBA cells were cultured in the absence of GM-CSF for 6 hours before the readdition of GM-CSF to their culture media for 0 to 60 minutes. Cells were harvested and equal-protein aliquots (30 μg) were immunoblotted with phospho–Stat-5. After stripping primary antibody, Stat-5 levels were determined by immunoblotting. (B) Mo7e cells were incubated with IL-3 for 30 minutes or pre-incubated with STI-571 for 60 minutes before IL-3. Cell lysates were analyzed for Stat-5 activation and Stat-5 levels by immunoblotting. STI-571 did not affect IL-3–mediated activation of Stat-5 in Mo7e cells. (C) MBA cells were left untreated (lanes 1-3) or were treated with 5 μM STI-571 (lanes 4-6) for 60 minutes before treatment with 500 U/mL GM-CSF or 30 ng/mL recombinant IL-3 (R&D Systems) for 15 minutes. Cell lysates were analyzed for bcr-abl and Stat-5 activation (phospho–Stat-5) and were compared to Stat-5 protein levels by sequential immunoblotting. (D) K562 cells were treated as described above and analyzed for changes in Stat-5, bcr-abl, and MAPK activation by immunoblotting (phospho–Stat-5, phospho-MAPK). Akt protein levels were unchanged by treatment and were used as a protein-loading control.