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Fig. 8. (A) Primary culture of embryonic (E11.5) endocardial cells (ECCs). ECCs form a colonized monolayer surrounded by fibroblastic-like OP9 feeder cells at passage 1 (p1). At p3, ECCs exhibit uniform, endothelial-like morphology with a typical `cobblestone' appearance. Approximately 80% of the ECCs expresses nuclear localized Nfatc1 (green; negative cells are indicated by arrowheads in DAPI staining). Negative control (no primary antibody) is shown in ECC(-Ab). (B) The 781 bp enhancer region. Two short stretch conserved sequences, ECE1 and ECE2, are shown with Nfat-binding sites highlighted in red and deleted GG or CC bi-nucleotides of core binding site are underlined. The arrows indicate the location of primers for the ChIP assays. (C) EMSA demonstrating that mutation of the Nfat-binding sites in either the N1 or N2 regions results in attenuation of Nfatc1 binding (arrows). (D) Results of the ChIP assay document PCR amplification of the chromatin region encompassing ECE1 and ECE2 from wild-type ECCs following immunoprecipitation with an Nfatc1 antibody (7A6) (arrow) and absence of chromatin amplification without antibody or using cultured Nfatc1-null ECCs.

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