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. 1999 Nov 15;19(22):9747–9755. doi: 10.1523/JNEUROSCI.19-22-09747.1999

Fig. 5.

Fig. 5.

Direct interactions between Sox10 and the β4 promoter region are functionally relevant. A, Sequences of the oligonucleotides used in electrophoretic mobility shift assays are shown. Mutations in the CA box oligonucleotide areunderlined. The sequence of an oligonucleotide corresponding to a consensus binding site for Sox proteins (Pevny and Lovell-Badge, 1997) is also shown. B, Electrophoretic mobility shift assays were performed using 5 fmol of a radiolabeled oligonucleotide (β4 in A) corresponding to nucleotides −82 to −48 of the β4 gene and 1 μg of T7-tag–Sox10 protein. The left lane shows DNA–protein complex formation in the absence of competitors. Competition experiments were performed using 100- and 500-fold molar excesses of competitor oligonucleotides (either unlabeled wild-type or mutated β4 oligonucleotides or the consensus Sox-binding site oligonucleotide). The right lane contains unbound β4 probe in the absence of Sox10 protein. C, Electrophoretic mobility shift assays were performed using 5 fmol of a radiolabeled oligonucleotide (CA Box in A) and 3.5 μg of rat brain nuclear extract. The left lane shows DNA–protein complex formation in the absence of competitors. Competition experiments were performed using 100-fold molar excess of competitor oligonucleotides (either unlabeled wild-type or mutated CA box oligonucleotides or the consensus Sox-binding site oligonucleotide). The right lane contains unbound CA box probe in the absence of protein. D, The mutations shown in A (Mutant CA Box) were introduced into the β4/luciferase construct pX1B4FH to create pX1B4FHmut. Wild-type pX1B4FH (FHwt) and pX1B4FHmut (FHmut) were cotransfected into Neuro2A cells with pCMVSox10. As controls, wild-type pX1B4FH was also transfected either alone (FHwt) or with pCMV5 (pCMV). Luciferase values were normalized to β-galactosidase expression as driven by the SV40 promoter. Fold induction was calculated relative to the normalized luciferase activity obtained by transfecting wild-type pX1B4FH alone. Error bars represent SDs. CAm, Mutant CA box.