Confocal microscopy analysis of NF-κB p65, STAT3, and STAT5 nuclear localization in unprimed and pro-NK-committed CB progenitors induced by SMFmb-IL-15. (A) CB hematopoietic progenitors were analyzed soon after immunopurification (day 0, panel A) or after 5 days in the presence of r-SCF/r-Flt3-L (day 5, panel B). Overlay pictures (original magnification, ×63). Left panels show basal conditions; central panels, 30 minutes of contact with SMF at 37°C; and right panels, 60 minutes of preincubation with 10 μg/mL neutralizing anti-IL-15Rα mAbs, followed by 30 minutes with SMF. CB progenitors were labeled with anti-NF-κB p65, anti-pSTAT3, and anti-pSTAT5 antibodies, and then incubated with Alexa Fluor488-GARa (goat antirabbit) antibody (green staining). Propidium iodide stained the nuclei red. Nuclear p65, STAT3, and STAT5 are indicated by nuclear yellow staining. As negative controls, cells were incubated with only the second reagent and propidium iodide. These data are representative of 3 different experiments. Images were obtained with a 63 ×/1.30 objective lens. (C) Western blot analysis of STAT5 phosphorylation using anti-pSTAT5 Ab in nuclear extracts of 5-day-old CB pro-NK progenitors stimulated or not by 45 minutes of contact with SMFmb-IL-15.