Figure 2. .
Functional consequences of the p.Val92Leu (V92L) mutation. A, Amino acid sequence comparison of the region immediately preceding the HMG domain of SOX10 proteins across evolution and of SOX subgroup E proteins (human sequences). Gaps are indicated by blanks. The first part of the HMG domain is boxed, and the amino acid substitution V92L from patient 3 is indicated by an arrow. SOX10 sequence reference numbers: human (accession number NP_008872.1), mouse (XP_128139.4), chicken (NP_990123.1), fugu (NP_001072112.1), zebrafish (NP_571950.1), human SOX8 (NP_055402.2), and human SOX9 (NP_000337.1). B, Subcellular localization of wild-type SOX10 and p.Val92Leu mutant. HeLa cells were transfected with pECE-SOX10 (WT) or mutant (V92L) constructs for 24 h, were fixed, and were immunostained for SOX10. Cultures were counterstained with DAPI. C and D, Transactivation capacities of SOX10 proteins. The MITF promoter (pMITF) (C) or the GJB1 (pCx32) promoter (D) luciferase reporters were transfected in Hela cells in combination with wild-type (WT) or mutant SOX10 proteins (V92L, E189X, 482ins6, and Y313X), and/or PAX3 (C) or EGR2 (D). Reporter-gene activations are presented as fold induction relative to the empty expression vector (pECE). Results represent the mean±SEM from six experiments, each performed in duplicate. Insets in C and D show a schematic representation of relative localization of SOX10 (white boxes), PAX3 (striped circles) or EGR2 (striped triangles) binding sites on MITF or GJB1 (Cx32) promoters.