Figure 4.

Nuclear extracts from LβT2 cells treated with the indicated concentrations of GnRH1 for 1 h (A) or 10⁻⁷ m GnRH1 for the indicated periods of time (in hours) (B) were incubated with a radiolabeled double-stranded oligo-probe corresponding to −126/−94 of the human FSHB promoter. DNA-protein complexes were resolved by native PAGE and visualized by autoradiography. C, Gel shifts were performed as described in A and B with nuclear extracts from LβT2 cells treated with 10⁻⁷ m GnRH1 for 1 h and the radiolabeled −126/−94 probe. Binding was competed with 20, 100, 200, or 1000-fold excess unlabeled probes corresponding to −126/−94 (lanes 4–7), −93/−61 (lanes 8–11), −60/−27 (lanes 12–15), or −26/+7 (lanes 16–19). D, Competition (comp) experiments in gel shifts were performed as described in C with the indicated WT (lane 3) and mutant −126/−94 probes (lanes 4 and 5). E, Competition experiments were performed as in D with WT (lanes 3 and 6) and mutant −93/−61 or −60/−27 probes (lanes 4, 5, or 7).