Determination of hemoglobin and globin chain expression. (A-C) Cation exchange and (D-F) reverse-phase chromatography analyses of cells cultured in (A,D) low-HbF versus (B,E) high-HbF culture conditions. The major peaks are labeled on each graph (y-axis, absorbance; x-axis, elution time). (C) The HbF/HbF + HbA ratios expressed as a percentage (y-axis) from 9 separate donor-matched erythroblasts cultured for 14 days in low-HbF (□) and high-HbF (■) conditions. Average values (Ave) are also shown. (F) ^Gγ/^Aγ globin ratios (y-axis) correspond to the results in panel C. *P < .05. (G) Western blot analyses were performed using total protein extract (25 μg/lane) from erythroblasts cultured in low-HbF (L) and high-HbF (H) conditions and harvested on days 2, 4, 6, 8, 10, 12, and 14. Antibodies to ζ-globin chain (ζ), ϵ-globin chain (ϵ), and GAPDH were used for detection. A vertical line between d6 and d8 was inserted to indicate the transition between 2 blots.