Temporal changes in expression of the C/EBP-ϵ isoforms during eosinophilopoiesis. CD34⁺ progenitors were differentiated to the eosinophil lineage by suspension culture in SCF, IL-3, IL-5, GM-CSF, and Flt3-L for 3 days, followed by only IL-3 and IL-5 thereafter. The cells were maintained at 0.5 × 10⁶ cells/mL, total and eosinophil counts determined every 3 to 4 days, and total RNA for RT-PCR and total protein for Western blotting prepared from 1 × 10⁶ cells. Cell proliferation (A) and the percentage of eosinophils (B) developing in the cultures based on differential cell counts using Fast Green/Neutral Red staining to distinguish secondary granule formation is shown. Semiquantitative RT-PCR was performed using α-³²P-dCTP and C/EBP-ϵ isoform selective primers, with β₂M amplified as the internal control for mRNA (cDNA) input (C). C/EBP-ϵ isoform protein expression was analyzed by Western blotting of whole cell lysates using a combination of anti–C/EBP-ϵ C-terminal (C-22; SC-158) and N-terminal (H-75; SC-25770) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), compared with GAPDH expression as the loading control (D). Representative results from 2 independent experiments are shown.