Fig. 6. FOS-1 can directly bind to egl-13 URS as a heterodimer with JUN-1, which is also expressed throughout the uterus at cell specification. (A) EMSA with labeled 100 bp homologous template containing the Fos/Jun binding site. Left gel: No band shift was detected with only lysate present (, lane 1), FOS-1 alone (lane 2), or JUN-1 alone (lane 3). A shifted complex (bottom arrow) was observed in the presence of both FOS-1 and JUN-1 (lane 4) and also with a Myc-tagged version of FOS-1 with JUN-1 (lane 5). Addition of polyclonal anti-Myc antibody in lane 6 produced a supershift due to increase in molecular mass of DNA-protein complex plus Ab (top arrow) and also completely neutralized the original band shift (bottom arrow) due to titration of protein from forming a stable complex. Right gel: The supershift was significantly reduced in the presence of unlabeled (COLD) sequence containing the egl-13 Fos/Jun binding site (lanes 4-6) but not effectively reduced by an excess of unlabeled sequence carrying a mutated Fos/Jun binding site (lanes 7-9). Gray triangles above lanes 4-6 and 7-9 represent increasing amounts (5x, 25x and 125x, respectively) of unlabeled 30 bp competitor oligonucleotides. (B) This diagram represents the position of the isolated 5.3 kb genomic sequence in pJUN-d/c::GFP in the context of the entire jun-1 locus. The NruI site is a starting reference point for 5 kb upstream of the first transcript. The translational starts for verified isoforms are indicated. (C-F) Uterine JUN-1 expression. (C,D) In the medial plane, expression of pJUN-d/c::GFP is detected in the AC (black arrowhead) as well as throughout the dorsal and ventral uterus. (E,F) In a lateral plane, VU intermediate precursors (white arrowheads) also express the translational reporter. Scale bar: 10 µm.