STAT5 expression and activation during eosinophil differentiation.

(A) Protein lysates were prepared from CD34⁺ cells, and cells differentiated toward eosinophils for 7, 10, 14, and 17 days. Western Blot analysis was performed with an N-terminal antibody against STAT5 (top panel), an antibody against tyrosine-phosphorylated STAT5 (middle panel), and as a control for equal loading with an antibody against β-actin (bottom panel). The experiment shown is representative of 3 additional experiments. (B) Nuclear extracts were prepared from differentiating eosinophils at days 7, 10, 14, and 17. Nuclear extracts were analyzed by EMSA using a ³²P-labeled β-casein probe. Competition experiments were performed with an unlabeled β-casein probe in lane 6 and with a nonspecific probe in lane 7. Supershift analysis was performed with nuclear extracts prepared from differentiating eosinophils stimulated with IL-5 for 15 minutes, with antibodies against STAT5, STAT3, and STAT1, respectively (lanes 9-11). The experiment shown is representative of 3 individual experiments.