Fig. 5

GATA4 is directly associated with the Barx1 promoter in mice. a The information of two binding sites with high scores predicted by computational analysis was chosen for further investigation. The matched sequence of GATA4 to Barx1 promoter was acquired from the JASPAR database. b The upper part corresponds to the structure diagram of the Dual-Luciferase reporter vector (pEZX-FR03). The sequences of the putative GATA4-binding sites in WT and mutants are shown below. c Quantitative results of dual-luciferase reporter assay (n = 5). “+” or “−” indicated the presence or absence of the reagent in dual-luciferase reporter assay. d The nuclear extracts from NCCs were analyzed for binding of GATA4 to Barx1 promoter using EMSA. Binding of GATA4 to biotin-labeled DNA probes is shown as “GATA4-Barx1 complex”. “+” or “−” indicated the presence or absence of the reagent in EMSA. The amount of the unlabeled fragment (cold probe and mutant probe) added in the competition assay was 300-folds of the amount of the labeled probe. A supershift band was shown when the nuclear extract was pre-incubated with 2 μg GATA4 antibody, which confirms the presence of GATA4 in both bands. e, f The quantitative PCR and ChIP gel shift assay for GATA4 binding to Barx1 promoter in NCCs. Lane 1, DNA marker; Lane 2, ChIP sample with GATA4 antibody; Lane 3, Input amplified by GATA4 primers; Lane 4 ChIP sample with IgG antibody; Lane 5, ddH₂O amplified by GATA4 primers. In quantitative PCR, y-axis indicated fold of enrichment normalized to control immunoglobulin. The data are presented as the mean ± S.E.M. from at least three independent experiments. ^**P < 0.01. ChIP chromatin immunoprecipitation, ddH₂O double-distilled water, EMSA electro-mobility shift assay, GAPDH glyceraldehyde 3-phosphate dehydrogenase, IgG immunoglobulin, NCC neural crest cell, WT wild type