Figure 4. The HD of Nkx6.1 interacts with the G1 element of the glucagon gene promoter.

(A) Schematic representation of the 5′-flanking region of the rat glucagon gene promoter depicting the control elements (G1–G5). The sequence of G1 is shown as well as the binding sites for Pax6, Cdx2/3 and Maf (bold nucleotides). The various probes used in EMSA are depicted and underlined nucleotides represent mutated sequences. Studies of Nkx-6.1 binding to the G1 (−100/−49) and G3 element (−274/−234) are represented in (B) and (C) respectively. Specific interactions of Nkx6.1 on glucagon probes was determined using an anti-Nkx6.1 antibody. pBAT-12 represent nuclear extracts derived from cells transfected with control empty vector. Competition assays were performed with increasing amounts of non-labelled probes ranging from 50- to 200-fold excess for native oligonucleotides and 200-fold excess for mutated oligonucleotides. SS, supershift.