Analysis of Stat5 function in human CD34⁺ cells via c-Kit signaling. (A) Purified human CD34⁺ cells from umbilical cord blood were stimulated with recombinant SCF (10 ng/mL) or IL-3 (10 ng/mL) for 30 minutes. Tyrosine phosphorylation of Stat5 was evaluated by Western blot analysis using an anti–P-Y-Stat5 antibody. SCF-mediated activation of Stat5 in CD34⁺ cells was also analyzed by immunocytochemistry with an anti–P-Y-Stat5 (AX1) antibody. (B) Human CD34⁺ cells were transduced or not (PBS) with the different TAT-Stat5 proteins (10 nM) for the indicated times. After extensive washes, the presence of recombinant TAT-Stat5 proteins in CD34⁺ cells were analyzed by Western blotting with the indicated antibodies. (C) CD34⁺ cells (50 × 10³; n = 4) were cultured in the presence of SCF (10 ng/mL) for 20 days. TAT-Stat5 proteins (10 nM) were added or left away (PBS) every 2 days in culture and cells were enumerated every 5 days. (D) Transduced cells grown for 30 days in the presence of SCF were analyzed for expression of FcϵR1 by flow cytometry. Immunocytochemical analysis (IC) was also performed on TAT-cS5^F protein-transduced cells with an antitryptase antibody. (E) The presence of metachromatic granules was detected after staining with toluidine blue. Results shown are representative of 4 independent experiments.