FIG. 7.
Inhibition of the nuclear export activity of Sox10 leads to decreased transactivation. (A) Luciferase assays after cotransfection of a luciferase reporter carrying the Sox10-responsive P0-promoter and expression plasmids for GFP (-), wild-type GFP-Sox10, and its mutants. Error bars, standard deviations. (B and C) Western blot analysis with an anti-Sox10 antibody (B) and electrophoretic mobility shift analysis using probe C/C′ (C) of whole-cell extracts from cells expressing the indicated proteins. The free probe is marked with an asterisk. The arrow indicates the complex between the oligonucleotide and GFP-Sox fusion proteins. (D) Comparison of DNA-binding affinity of wild-type Sox10 (WT) and Sox10L138A (L138A) mutant. Electrophoretic mobility shift analysis was performed using labeled C/C′ as probe and increasing molar excess of unlabeled C/C′ as competitor. The signal intensity was quantified using the IPLab software package. (E) Luciferase assays after cotransfection of a luciferase reporter carrying the Sox10-responsive P0 promoter and expression plasmids for GFP-Sox10 or GFP (-) in the presence or absence of LMB. The data were normalized to cotransfected β-Gal under control of the cytomegalovirus promoter. Error bars, standard deviations. (F and G) Western blot analysis with an anti Sox10 antibody (F) and electrophoretic mobility shift analysis using probe C/C′ (G) of whole-cell extracts from LMB treated or untreated cells expressing GFP (-) or GFP-Sox10. (H) Semiquantitative RT-PCR analysis of endogenous P0 and PLP transcript levels in N2A Tet-On cells capable of inducibly expressing Sox10. Cells were treated with doxycycline for 48 h (+DOXY) or doxycycline for 48 h and LMB for 24 h (+DOXY +LMB) to induce Sox10 expression. cDNA from uninduced cells served as control.