FIGURE 8.

Endogenous Pias proteins bind to the Lef-1 promoter. A, schematic representation and location of the Pitx2 binding site in the Lef-1 promoter. # indicates the Pitx2 binding element (TAATCC). B, ChIP of endogenous Piasy binding to the Pitx2 element within the Lef-1 promoter in LS-8 cells. Rabbit antisera used as a control IP and Piasy antisera from Santa Cruz were used to IP Piasy binding to the chromatin. We have previously shown Pitx2 binding to this element (33). The input chromatin is shown as a positive control for the ChIP. C, control ChIP using the Piasy antisera and primers to a 4.7-kb upstream region of the Lef-1 promoter. This chromatin does not contain a Pitx2 binding site and was not IPed using Piasy antisera, the primers did amplify the input chromatin. D, ChIP of endogenous Pias1 binding to the Pitx2 element within the Lef-1 promoter in LS-8 cells. Rabbit antisera was used as a control IP and Pias1 antisera from Santa Cruz was used to IP Pias1 binding to the chromatin. The input chromatin is shown as a positive control for the ChIP. E, control ChIP using the Pitx2 antisera and primers to an upstream region of the Lef-1 promoter. This chromatin does not contain a Pitx2 binding site and was not IPed using Pias1 antisera, the primers did amplify the input chromatin. F, ChIP DNA fragment concentrations were evaluated by real-time PCR to calculate the fold-enrichment of Pias1 and Piasy antibody IP group, respectively. Both IP enrichments were normalized to the IgG group.