c-Src-kinase–dependent STAT5 activation regulates IL-3–induced ex vivo expansion of angiogenic cells. (A) CD45⁺ cells cultured with or without IL-3 were analyzed by Western blot (WB) using anti-p-STAT5 or anti-STAT5 antibodies. (B) Cell extracts from IL-3-cultured CD45⁺ cells, pretreated or not with AG-490, PP1, PP2, and SU6656, were subjected to SDS-PAGE. The filter was IB with anti-p-STAT5 and anti-STAT5 antibodies. (C) CD45⁺ cells, cultured as indicated, were evaluated for c-Src kinase activity as described in “Antibodies” (*P < .05, EBM-2 + IL-3 vs EBM-2 − IL-3 or vs EGM-2; §P < .05, EBM-2 + IL-3 + SU6656 vs EBM-2 + IL-3). The amount of c-Src in cell extracts was evaluated by WB using a specific antibody. (D) IL-3-cultured CD45⁺ cells transfected with STAT5A and/or STAT5B siRNA or with the scrambled sequence (scramble) were lysed. The filters were IB with anti-STAT5, anti–cyclin D1, or anti–β-actin antibodies. (E) Q-RT-PCR was performed to evaluate the expression of arterial markers in CD45⁺ depleted or not (scramble) of STAT5A and/or STAT5B as indicated. Expression levels are presented as above described. (*P < .05, experimental groups vs scramble.) Three independent experiments were performed with comparable results. IL-3–treated EC were used as positive control (+) throughout the figure.