Western blot and confocal microscopy analysis of STAT6 phosphorylation and nuclear localization in committed CB progenitors induced by r-IL-15 and SMFmb-IL-15. CB progenitors were analyzed after 5 days in the presence of r-SCF/r-Flt3-L (pro-NK commitment) or in Stemα AE medium (proerythroid commitment). (A) Western blot analysis of STAT6 phosphorylation using anti-pSTAT6 Ab in CB proerythroid progenitors incubated or not (basal) with 0.1 and 100 ng/mL human r-IL-15. Membrane was reprobed with antibody recognizing the native protein. Confocal microscopy of pro-NK (B) and proerythroid cells (C) treated with r-IL-15. Overlay pictures (original magnification, ×63). Panels Bi and Ci indicate negative control; panels Bii and Cii, basal conditions; panels Biii and Ciii, 30 minutes of incubation at 37°C with 0.1 ng/mL human r-IL-15; and panels Biv and Civ, 30 minutes of incubation at 37°C with 100 ng/mL human r-IL-15. Confocal microscopy of pro-NK (D) and proerythroid cells (E) in contact with SMFs. Overlay pictures (original magnification, × 63). Panels Di and Ei indicate negative control; panels Dii and Eii, basal conditions; panels Diii and Eiii, 30 minutes of contact at 37°C with SMF; and panels Div and Eiv, 60 minutes of preincubation with 10 μg/mL neutralizing anti-IL-15Rα mAbs, followed by 30 minutes of contact with SMFs. As negative controls, cells were incubated with rabbit IgG, the second reagent, and propidium iodide. These data are representative of 3 different experiments. (F) Western blot analysis of STAT6 phosphorylation using anti-pSTAT6 Ab in nuclear extracts of 5-day-old CB pro-NK progenitors stimulated or not by 45 minutes of contact with SMFmb-IL-15. Images were acquired with a 63 ×/1.30 objective lens.