Analysis of transcription factor phosphorylation by Western blotting in human CD34⁺ cells from various sources. Hematopoietic progenitors isolated from BM (A), MPB (B), and CB (C) were incubated or not (basal) with neutralizing anti—IL-15 mAb for 1 hour. BM (D) and CB (E) CD34⁺ cells were also incubated with neutralizing anti—IL-15Rα, IL-15Rβ, or anti—IL-6R gp130 (isotype-matched control) mAbs for 1 hour. Cell extracts were analyzed by Western blotting using anti—phospho-STAT3 (pSTAT3), -STAT5 (pSTAT5), and -IκBα (pIκBα) antibodies (upper blots). Each membrane was reprobed with antibodies recognizing the native proteins or the β-actin (lower blots), showing equal amounts of protein in each sample. These data are representative of 3 different experiments.