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Fig. 4. Selective persistence of NACA production during erythroid versus megakaryocytic or granulocytic differentiation of CD34+ cells. (A) Western-blot analyses were performed on CD34+ cells: (0) immediately after immunomagnetic purification; (2) after culture for 2 days; (6) after six days under expansion conditions. Alternatively, the western blot was performed after culture under conditions optimized for the cell growth and maturation of: erythroid (days 7+2, 7+5, 7+7, where day 7 corresponds to the end of the first Epo-free phase of the culture; Epo was added at day 7 and additional day numbers correspond to the duration of culture in the presence of Epo); megakaryocytic (days 6, 7, 10, 12, 14); granulocytic (days 6, 7, 14). Cells were harvested at the indicated times of culture. 20 µg total protein extracts were separated by 9% SDS-PAGE and analysed by western blot. Membranes were probed with a polyclonal immune serum against NACA and then probed again with an antibody against the ubiquitously expressed Grb2 adaptor. A result typical of those obtained from three different cell cultures is shown. Arrows indicate the molecular-weight markers. (B) Flow-cytometry analysis of CD34, GPA, CD42b, CD61 and CD15 surface-marker expression on cells either immediately following CD34+ purification (D0) or after 6 days of culture (D6) in expansion conditions, or after 10 days of culture in conditions optimized for erythroid, megakaryocytic or granulocytic growth and maturation, respectively. Bold profiles represent the fluorescence distribution of an isotype-matched control antibody. (C) Quantitative real-time RT-PCR analysis of NACA expression performed on mRNA from CD34+ cells either after 3 days or 6 days of culture under expansion conditions or after 10 days of culture under conditions optimized for erythroid, megakaryocytic and granulocytic growth and maturation, respectively. The expression of NACA transcript is expressed as a ratio relative to the expression of GAPDH.
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