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. 2003 Mar 17;197(6):763–775. doi: 10.1084/jem.20020150

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Copyright © 2003, The Rockefeller University Press

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Figure 6. — IL-15 signal transduction: IL-15R/GM-CSFR cross talk in CB^Pr CD34⁺/CD56⁻ cells. Analysis of STAT signal transduction by Western blotting. CB CD34⁺ cell populations were expanded by incubation for 5 d in the presence of SCF and Flt3-L. These cells displayed a CD34⁺/CD56⁻ phenotype with no expression of lineage-specific markers. These CB^Pr CD34⁺/CD56⁻ progenitors were incubated with 10 ng/ml rIL-15 or rGM-CSF for 15 min at 37°C. Sister cultures were pretreated for 1 h with neutralizing anti-IL-15Rβ/γc, anti-GM-CSFRβ, or anti-IL-6R gp130 mAbs. Cell extracts were analyzed by Western blotting with anti-phospho-STAT3 (pSTAT3), anti-phospho-STAT5 (pSTAT5), and anti-phospho-STAT6 (pSTAT6) antibodies. Membranes were then reprobed with antibodies recognizing the native proteins. To correct for possible variations in the amount of protein loaded, values are expressed as pSTAT/STAT ratios. pSTAT/STAT levels were determined by densitometry, including correction for background (NIH Image software). Results are expressed as increases with respect to untreated cells. The data presented are representative of two independent experiments.