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. 2007 Oct 8;27(24):8571–8582. doi: 10.1128/MCB.01350-07

FIG. 5.

FIG. 5.

Sp3 binds to the mouse Carp promoter in vitro and in vivo. (a) The Carp promoter region. The five Sp motifs (Sp C1 to Sp C5) are indicated, as are the Nkx2.5 and GATA motifs. (b) Gel retardation assay. The specificity of binding to the Sp C1 to Sp C5 oligonucleotides was analyzed through the addition of a 50-fold molar excess of a competitor oligonucleotide containing two Sp sites (18). Supershifts were performed to analyze the protein complexes with antibodies against Sp1 or Sp3. The arrows point to the different protein complexes formed; note that there are several isoforms of Sp3 (46). (c) ChIP reactions were performed on formaldehyde-cross-linked chromatin isolated from E14.5 ventricles, using the antibodies indicated. Enrichment for Carp promoter sequences was determined by real-time quantitative PCR, using amplification of an unrelated fragment of the Gata2 gene as a standard to normalize for the amount of template DNA in the reactions. (d) Real-time PCR analysis of Carp mRNA expression in E14.5 hearts of WT and Sp3 knockout (Sp3−/−) embryos. Expression of cyclophilin A mRNA was used to normalize for the amount of RNA. Error bars indicate standard errors of the means. The average Carp expression level in WT hearts was set to 100.