Figure 2.

Hes-1 directly binds the N-boxes of the GluR1 promoter in vitro. A, EMSA assay of N2N3 probe that contains two N-boxes was labeled with γ-³²P. Sequences for wild-type probe and mutant probe are shown at the bottom. The capital letters indicate the sequence for N-box. The underlined capital letters represent the mutated residues. m, Mutation. Each wild-type probe and mutant probe was incubated with His-Hes-1 that is purified from E. coli. Lane d showed that His-Hes-1 bound the N2N3 probe directly. Lanes a–c showed the binding signal of N-box double mutant N2N3 probe (N2mN3m), mutant N2 probe (N2mN3), and mutant N3 probe (N2N3m) (CACAAG→CCATGG) with His-1. Lane e showed the result with the addition of 50× cold N2N3 competitor probe. Lanes f–h revealed that addition of 50× mutant cold competitor (N2mN3, N2N3m, or N2mN3m) partially decreased the binding signal, but cold N2mN3m probe cannot abolish the signal from the hot N2N3 probe. His antibody caused supershift of the band in lane i (indicated by the top arrow), but control IgG had no such an effect (lane j). Lanes m–p showed the result of probe only. The binding signal for the His-Hes-1-probe complex is also shown (indicated by the bottom arrow). The capital letters at the bottom indicate the sequences for individual N-boxes. The quantified result of binding is shown at the bottom panel. B, EMSA assay of different N-probes that contain individual N-box were labeled with γ-³²P. Sequences for each probe are shown at the bottom. The capital letters indicate the sequence for the N-box. Different N-probes were incubated with His-Hes-1 that is purified from E. coli. EMSA profile revealed that Hes-1 binds to all these N-boxes, and this binding was reversed by individual cold probe competition. The binding was further reversed by mutant cold probe competition. His antibody caused a supershift of the band (indicated by the top arrow). The binding signal for the His–Hes-1–probe complex is also shown (indicated by the bottom arrow). C, Results from ChIP assay followed by real-time PCR showing specific binding of endogenous Hes-1 to endogenous GluR1 promoter in cultured cortical neurons using the Hes-1 antibody and the primer for GluR1 promoter. Experiments are in duplicate (n = 3 for each group). Data are expressed as mean ± SEM. ^#p < 0.001.