rs12469063 alters a CREB1 transcription factor binding site. (A) Principle work flow for the identification of upstream binding factors by affinity chromatography and mass spectrometry. (B) CREB consensus sequence compared to the human sequence spanning rs12469063. (C,D) Creb/CREB and Meis1/MEIS1 were both detected in the mantle zone of the ganglionic eminences and show colocalization (bar in C represents 500 µm; bar in D represents 250 µm). (D) Magnification of rectangle in C. (E) Supershift EMSA assay with a specific antibody against CREB showed significant reduction of DNA-protein complex formation compared to unspecific IgG antibody (control) for overexpressed CREB1 in 293T nuclear cell lysate (lanes 1–6) and E12.5 forebrain nuclear extract (lanes 7–12, arrowhead). Complete abolishment of specific binding with an oligonucleotide, in which the entire CREB motif is deleted (lane 13). CREB consensus oligonucleotide shows specific binding, being absent with the mutated CREB consensus oligonucleotide (lanes 14,15). Arrow indicates additional allele-specific band.
