Analysis of phospho-STAT3 nuclear localization in human CD34⁺ cells from various sources by confocal microscopy. Hematopoietic progenitors isolated from BM (A), MPB (B), and CB (C) were incubated or not (basal) with neutralizing anti—IL-15 or anti—IL-15Rα mAbs for 1 hour and were analyzed for pSTAT3 expression and nuclear localization. Original magnification, × 63. The cells were permeabilized and labeled with anti-pSTAT3 antibodies, followed by incubation with Alexa Fluor488-GAR antibody. Propidium iodide stained nuclei in red, whereas phospho-STAT3 stained in green. As negative controls, cells were incubated with the second reagent and propidium iodide. Only nuclei were revealed (red staining). As a control for specificity, we used the IL-15—dependent CD34⁺ TF1β cell line (D), which displays high-affinity IL-15R (IL-15Rα/β/γc). These cells were treated for 1 hour with the neutralizing anti—IL-15, anti—IL-15Rα, or anti-gp130 subunit of the IL-6R (isotype-matched control) mAbs. Subsequently, they were incubated with recombinant IL-15 at 10 ng/mL and were analyzed for the nuclear localization of pSTAT3. STAT5 is not shown because anti—IL-15 mAb inhibits its phosphorylation. These data are representative of 3 different experiments.