Defining a competition between FOXO1 and POU5F1 (Oct4) for DNA binding at the RAB5A and ERMN loci. (A) Oligonucleotides originating from the RAB5A and ERMN upstream control regions that contain annotated octamer and FOXO1 sites were analyzed for FOXO1 or POU5F1 (Oct4) binding. (B) A radiolabeled oligonucleotide was incubated with recombinant POU5F1 (Oct4) or FOXO1 and fractionated by native PAGE. Antibody added prior to loading was used to supershift POU5F1 (Oct4) containing species (marked S). A panel of binding assays containing increasing amounts of POU5F1 (Oct4) was used to assay the ability of POU5F1 (Oct4) to displace FOXO1. (C) An analogous binding assay performed with increasing the amounts of FOXO1 was used to test binding competition at the ERMN loci. (D) POU2F1 (Oct1) and FOXO1 protein levels were measured in differentiated and undifferentiated extract. Binding competition between POU2F1 (Oct1) and FOXO1 was tested by gel shift on the RAB5A probe. Increasing concentration (1, 5, 10 μL) of FOXO1 was incubated with a fixed concentration of POU2F1 (Oct1). The ratio of multiply bound to monomeric probe was quantified by phosphoimager.
