Western blot analysis of transcription factor phosphorylation in TF1β (IL-15Rαβγc) and M07Sb (IL-15Rβγc) cells after short-term contact with SMFs. Leukemic TF1β (A) and M07Sb (B) progenitors were untreated (basal) or incubated with SMFs for 30 minutes. Parallel cultures were preincubated with neutralizing anti-IL-15Rα, IL-15Rβ, or anti-IL-15 mAbs for 1 hour and then with SMFs for 1 hour. Leukemic progenitors were then detached from myofibroblasts by gentle shaking and centrifuged with 10 mL of growth medium. Cell extracts were analyzed by Western blotting using anti-IκBα (pIκBα), anti-phospho-STAT3 (pSTAT3), anti-phospho-STAT5 (pSTAT5), and anti-phospho-STAT6 (pSTAT6) Abs (top blots). Each membrane was reprobed with antibodies recognizing the native proteins or β-actin (bottom blots). β actin was used as a loading control. These data are representative of 3 different experiments. Flow cytometry analysis of intracellular Bcl-X, cyclin D1, and SOCS3 expression in permeabilized 5-day-old CD34⁺ CB progenitors (C) and MO7Sb cells (D). Cytokine-deprived cells were incubated for 4 hours with the JAK2/STAT5 inhibitor AG490, and then control and treated cells were cocultured for 24 to 48 hours with SMFs. These data are representative of 3 different experiments. In C and D, gray line indicates IgG; black line, Basal; red line, SMF; blue line, AG490+SMF.