Influence of IFN-α on IL-7R proximal and distal signal transduction events. (A) IFN-α has no effect on IL-7–induced phosphorylation of STAT5. MACS-enriched CD34⁺ thymocytes were preincubated overnight in culture medium only or in culture medium with IFN-α (1000 U/mL). The following day, cells were stimulated for 15 minutes with IL-7 (1 ng/mL), IFN-α (1000 U/mL), or both, stained for intracellular phospho-STAT5, and analyzed by flow cytometry. Shaded curves represent isotype controls; open curves, specific staining. Numbers indicate MFI of specific stainings. (B) IFN-α does not prevent IL-7–mediated down-regulation of p27^kip1 protein or hyperphosphorylation of Rb. MACS-enriched CD34⁺ thymocytes were cultured for 48 hours with IL-7 in the absence or presence of IFN-α (5000 U/mL). Total cell lysates were analyzed by Western blot analysis using antibodies against either p27^kip1 or Rb. Actin blotting was performed to ensure equal protein loading. Results are representative of 2 independent experiments.