Scl regulates NF-E2 expression in platelets. (A) Quantitative PCR on platelet cDNA. Platelets were purified from mice of the indicated genotypes and used to generate cDNA, which was amplified by real-time PCR using primers specific for the indicated genes. (B) Partial sequence of the NF-E2 internal 1b promoter. Nucleotides 420-652 of the human promoter/exon 1b region are shown. E boxes present in the region are shown in bold. Tandem GATA motifs, which have been shown to be important in regulation, are underlined. (C) Chromatin immunoprecipitation using Meg-01 cells. Cell extracts were prepared from the human megakaryoblastic-cell line Meg-01 and either 10% input (input), or immunoprecipitates using the indicated antibodies, were used to PCR amplify the region of the NF-E2 promoter/exon 1b sequence shown in panel C. “None” indicates no antibody; control Ig, normal rabbit serum. (D) Chromatin immunoprecipitation using Meg-01 cells. As in panel C, using an anti–GATA-2 antibody as indicated. (E) Chromatin immunoprecipitation using K562 cells. Cell extracts were prepared from the human erythroleukemic-cell line K562 and subjected to chromatin immunoprecipitation as in panel C. (F) Transactivation of the NF-E2 promoter. A luciferase reporter construct containing the 232-bp NF-E2 promoter region as in panel B was transfected into 293T cells along with expression plasmids for the indicated genes. The activities shown are normalized to a cotransfected Renilla luciferase expression plasmid. Data are mean + SD of triplicate determinations.