Fig. 3. Mutations in XSOX3. (A) The HMG box (pfam00505.6) is characterized by three -helices and nine conserved residues. In this image, generated using the Cn3D viewer, the N- and C-termini are indicated and conserved residues are marked in red. (B) The conserved amino acids of the HMG box are indicated using the single-letter amino acid code. With the exception of m68, which is outside the conserved core region, the mutations we generated in the XSOX3 HMG box are indicated. (C) The sequence of the XSOX3 HMG box is displayed and the mutations generated for this analysis are indicated. The first residue of this sequence, D, corresponds to amino acid 38 of the full-length XSOX3 sequence. (D) XSOX3-V5H₆ polypeptides (wild type, m7, m8, m17, m40, m55 and m68) generated by in vitro transcription/translation (TnT) were analysed by SDS-PAGE/immunoblot using the anti-V5 antibody. All accumulated to similar levels (arrow). The nature of the extraneous bands (*) are not known. (E) TnT-synthesized proteins were used in oligonucleotide gel-shift studies with the DC5 SOX-binding oligonucleotide. Unprogrammed lysate (Un) showed no shift and no effect upon the addition of antiV5 antibody (+). Oligonucleotide gel shift and antibody-induced supershift were observed upon addition of XSOX3 (wt) and m55 (m55) polypeptides, but not with m7, m8, m17, m40 or m68 polypeptides.