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Figure 1 | BMC Developmental Biology

Figure 1

From: Er81is a downstream target of Pax6 in cortical progenitors

Figure 1

Pax6 bind specifically to a putative Er81 promoter. (A) The schematic depicts the putative Er81 promoter, showing the relative positions of a potential Pax6-binding site (BS) and an external control sequence used for ChIP assays (see below). A comparison of the DNA sequences for the potential Pax6-binding site identified in the putative Er81 promoter (at position -1190-1225), the perfect Pax6-binding site (Cons), and the mutated site (Mut) is also indicated. (B) EMSA analysis of Pax6 binding to 32P-labeled probes corresponding to the potential Pax6 binding site, a perfect Pax6-binding site, and a mutated site. The arrows, open arrowhead and closed arrowhead indicate free probes, probe-protein complexes, and probe-protein-antibody complexes, respectively. Binding of Pax6 to its perfect Pax6-binding site (lane 3) and potential binding site in the putative Er81 promoter (lane 6) is clearly detectable. Probe binding is not observed using the TNT rat reticulocyte lysate (RRL) as a protein control (lanes 2, 5). Binding specificity was confirmed by pre-incubating with an anti-Pax6 antibody, which resulted in a super-shifted DNA-Pax6 protein-Pax6 antibody complex (lanes 4, 7). Binding of Pax6 was completely abolished by mutation of the Pax6 binding-site sequence in the Er81 promoter (lanes 9, 10). (C) ChIP assay. Pax6 antibodies precipitated chromatin containing the Pax6 binding site in the region -1322 to -1040 of the Er81 promoter (lane 2), but were unable to precipitate chromatin isolated from a region outside of the Pax6 binding site (lane 4). Preimmune serum (lane1, Pre) or GFP (lane3) antibodies failed to precipitate chromatin.

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