Expression of Id1 and Id2 is regulated during granulocyte differentiation. Umbilical cord blood–derived CD34⁺ cells were differentiated toward eosinophils or neutrophils in presence of the cytokines FLT-3 ligand, SCF, GM-CSF, IL-3 and IL-5, or G-CSF. After 3 days of culture, eosinophil progenitors were cultured in the presence of IL-3 and IL-5, whereas neutrophil progenitors were cultured in the presence of G-CSF alone after 6 days of culture. (A) Protein lysates were made immediately after isolation from cord blood and after 3, 7, 14, and 17 days of differentiation. Western blot analysis was performed with an antibody against Id1 (top blot), an antibody against Id2 (middle blot), and, as a control for equal loading, an antibody against β-actin (bottom blot). The experiment shown is representative of 3 independent experiments. (B) Protein levels were quantified using ImageQuant software (Amersham Biosciences, Freiburg, Germany), and data are depicted as an average of 3 independent experiments plus or minus the standard error of the mean (SEM). (C) RNA was isolated from cells differentiating toward eosinophils and neutrophils at the time points indicated. Quantitative real-time PCR reactions were performed using gene-specific primers for Id1 (left) and Id2 (right). Values were corrected for the expression of HPRT in all samples. The experiment shown is representative of 3 independent experiments.