Stat5 expression and activation.

Cells from AML patients in the group with normal IL-3Rα levels and patients exhibiting high IL-3Rα expression were incubated for 15 minutes at 37°C either in the absence (−) or in the presence (+) of IL-3. TF1 cells were used as a control for Stat5 activation by IL-3. (A) Total cell extracts (20 μg) were analyzed by EMSA using a specific radiolabeled oligonucleotide corresponding to the SBE motif present within the β-casein promoter. Supershift assay was performed after incubation of cell extracts with anti-Stat5 as indicated. Arrows indicate the supershifted band. (B) Thirty micrograms whole-cell lysates was separated on 7% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted sequentially with the indicated antibodies to evaluate Stat phosphorylation status (STAT-5Y) and protein content (STAT-5). These are representative EMSA and immunoblot experiments that were repeated 3 times with cell extracts from different AML patients.