Change in Stat5 isoform activation during GM-CSF–induced maturation of FdCP1 cells.

(A) FdCP1(c.19) cells were either cultured in WCM or differentiated in GM-CSF for 3 days. Cells were then starved (−) and stimulated with IL-3 (+; 10 ng/mL, 15 minutes). Nuclear extracts were evaluated by sequential immunoblotting with Stat5 phospho-specific (upper panel) and pan–Stat5-specific antibodies (lower panel). Recombinant inactive Stat5b (rStat5b) was included as a control (lane 5). The mobility of p96 (Stat5a), p94 (Stat5b), p80 (truncated Stat5b), and p77 (truncated Stat5a) are indicated in the left margin of each panel. (B) FdCP1(c.19) cells were cultured as in A. Differentiated cells, with increased forward and side scatter, were collected by flow cytometry to increase their purity to more than 95% (compare with Figure 3). CHAPS extracts were prepared and assayed on a recombinant FLAG-tagged Stat5b (rFlagSt5b) substrate prepared by overexpression in 293 cells.21 The mobility of rFlagSt5b (p94) and the cleaved product (p80) are indicated in the left margin of each panel.