Fig 2.

Analysis of Gata4 and Sp1 binding to the murine EpoR promoter. (A) Electrophoretic mobility shift assays demonstrate binding of Gata4 (probe nt-74/−45 GATA) and Sp1 (probe nt-44/−14 GC-rE) to DNA elements of the murine EpoR promoter. Asterisks indicate specific Gata4 and Sp1-mediated supershifts by antibodies directed against Gata4 (αGata4), Gata6 (αGata6) and Sp1 (αSp1). Of note, incubation with an antibody directed against Gata4 totally abolishes the specific complex generated by binding of Sp1 to the −44/−14 GC-rE. (B) Chromatin-immunoprecipitation assays with the EpoR minimal promoter were performed in freshly prepared cardiomyocytes (male C57/BL6). Immunoprecipitation with an antibody against Gata3 (αGata3) served as negative control, whereas that with the antibodies against RNA polymerase II (αRNA Pol II) and against acetylated histon 3 (αAcH3) as positive controls indicate active transcription of the EpoR gene. Precipitated DNA was amplified by PCR with primers spanning the EpoR promoter (nt-168/+161). Amplification of the Gapdh promoter served as control. Gel pictures were inverted.