Binding of FOG-1 and GATA-1 to β-chain promoter in BMMCs at different developing stages. (A) Expression profile of c-kit and FcϵRI analyzed by flow cytometry. 0W indicates freshly prepared Lin^- cells; 2W, immature BMMCs developed from Lin^- cells after 2-week culture; and 6W, mature BMMCs developed from Lin^- cells after 6-week culture. (B) Transcription levels of FOG-1 and β-chain during BMMC development. The mRNA levels of FOG-1 and β-chain in different developing stages of BMMCs and PT18 were measured by quantitative PCR and are represented as the ratio to that of Lin^- (0W). Data are represented as the average ± SD of triplicate samples. (C) FOG-1 and GATA-1 in vivo binding to β-chain promoter. Binding between β-chain promoter (-197/+128) and FOG-1 or GATA-1 was analyzed by ChIP assay using anti-FOG-1, anti-GATA-1, or each isotype control Ab. The GAPDH gene served as control. (D) Quantitative analysis of FOG-1 and GATA-1 binding to β-chain gene by ChIP assay using real-time PCR. ▪ indicates anti-FOG-1 Ab; ▨, goat IgG (control of anti-FOG-1 Ab); ▦, anti-GATA-1 Ab; □, rat IgG (control of anti-GATA-1 Ab). The results are expressed as mean ± SD for 2 PCRs with duplicate samples on each 2 independent ChIPs. Relative input units are calculated from Ct values as described in “ChIP assay.”