The Axl/Gas6 pathway is required for normal IL-15 signaling in human CD34⁺ HPCs. (A) CD34⁺ HPCs were isolated from human peripheral blood and treated with KL and FL for 5 days. Cells were washed, rested, and then treated with either irrelevant Fc or Axl-Fc in the presence of IL-15 for 30 minutes. Harvested cells were subject to Western blot to detect phospho-STAT5, and actin was used as a loading control. The figure is the representative of 2 similar experiments. (B) In a separate experiment, CD34⁺ cells were prepared and treated as described in panel A. Assessment of intracellular STAT5 phosphorylation using flow cytometry was performed while gating on CD34⁺ cells. Shown is 1 of 2 similar experiments. Dotted line represent media only; filled histogram, IL-15 + irrelevant Fc; open histogram, IL-15 + Axl-Fc. Dotted line and open histogram were indistinguishably overlapped.