Expression of NF-YA in response to SCF during erythroid differentiation. (A) Quantitative real-time PCR was performed to determine NF-YA expression levels for the indicated days after CD34⁺ cells were added with SCF (50 ng/mL) from day 6 to day 14. Results are shown as mean ± SD from 3 different donors. *P < .05 versus unstimulated cells. (B) CD34⁺ cells grown in EPO-containing medium for 6 days were incubated with SCF (50 ng/mL) for the indicated times, and whole cell lysates (30 μg) were analyzed by immunoblot with thioredoxin (Trx), Ref-1, or NF-YA antibodies. The lowest panel shows the same blot stripped and reprobed with anti–β-actin antibody to confirm that similar amounts of protein extracts were analyzed in each lane. (C) Immunofluorescence analysis of Trx and Ref-1 localization after cells grown in EPO-containing medium for 6 days were stimulated with SCF (50 ng/mL) for the indicated times. Representative immunoblots from 3 independent experiments are shown.