EMSA analysis of the hGATA and mGATA sites of GPIIb promoters.

(A) FACS analysis of erythrocytic (GPA) and megakaryocytic (CD41) cell surface antigen expression on CD34⁺ progenitor cells isolated from human umbilical cord blood and induced to differentiate into erythroid and megakaryocytic cells. After 12 days of culture, the cells were stained with FITC-antihuman GPA and PE-antihuman CD41 antibodies. Isotype-matched nonspecific antibodies were used as controls. Percentages of positive cells are indicated. (B) RT-PCR analysis of GPIIb expression. Total RNA was isolated from primary erythroid and megakaryocytic cells and from permanent cell lines (HEL, K562, LIN-175, and MEL). RT-PCR reactions were performed, including reverse transcriptase negative control (RT−) for each sample and H₂O blank for each oligonucleotide primer. Amplification of GAPDH and HPRT was performed on each human and murine cDNA sample respectively, as an internal standard. (C) GATA-binding activity analysis. Cell specificity of the human −463 (hGATA) and murine −456 (mGATA) sites were analyzed with nuclear extracts from human megakaryocytes and erythroblasts. Sequence comparison between hGATA (previously described8) and mGATA probes is reported. The GATA-1 consensus binding sites are underlined. An asterisk represents nucleotide mismatch between the 2 sequences. Position of the DNA-protein complexes in the EMSA experiments are indicated by arrows (A and B). (D) Cell specificity and supershift assays. The mGATA sequence binding activity was analyzed with human nuclear extracts from HEL, K562, HeLa, and murine nuclear extract from LIN-175 and MEL, in the absence (−) or the presence of the indicated antibody (right panel). Positive control indicating positions of the DNA-protein complexes (arrows) was obtained with the human probe and nuclear extract from erythroid cells, in the absence (band A corresponding to GATA-1 binding) and in the presence (band C, supershift) of anti–GATA-1 antibodies (left panel).