Figure 3.

DLX1 and DLX2 proteins specifically bind to Gad67/Gad1 promoter regions i and ii in situ. EMSA showed recombinant DLX1 or DLX2 binding to α-P³²-labeled (A) Gad67 promoter region i, and (B) Gad67 promoter region ii oligonucleotides containing candidate homeodomain binding sites (Fig. 1A, regions in purple boxes). GE nuclear-extracted proteins also bound to α-P³²-labeled oligonucleotides containing specific TAAT/ATTA binding motif for (C) the Gad67 region i second TAAT motif, and for (D) the Gad67 region ii fourth TAAT motif (Fig. 1A, motifs in purple boxes). A, B, Radioactive oligonucleotide probes were incubated alone (lane 1), with recombinant DLX1 proteins (lanes 2–5), with recombinant DLX2 proteins (lanes 6–9), with unlabeled competitive probes (lanes 3, 7), with specific DLX1 or 2 antibodies (lanes 4, 8), and with nonspecific antibodies (lanes 5, 9). C, D, radioactive oligonucleotide probes were incubated alone (lane 1), with GE nuclear extract (lanes 2–6), with unlabeled competitive probes (lane 3), with specific DLX1 antibody (lane 4), with specific DLX2 antibody (lane 5), with nonspecific antibodies (lane 6). A–D, Binding of DLX proteins to a specific oligonucleotide sequence results in a gel shifted band, indicated by solid arrows. Binding of DLX protein to a specific oligonucleotide and to a specific DLX antibody results in a gel supershifted band, indicated by broken arrows. (DLX1: unbold arrow, DLX2: bold arrow). r, Recombinant; 1, DLX1 protein or anti-DLX1 antibody; 2, DLX2 protein or anti-DLX2 antibody; I, irrelevant/nonspecific antibody.