Sox10 and Myrf interact physically and functionally.

(A) Co-immunoprecipitation (IP) of endogenous Myrf with anti-Sox10 antiserum (αSox10) or preimmune serum (PI) from OLN93 cell extracts. The upper panel shows western blot (WB) detection of Sox10, while the lower panel probes the presence of Myrf in the precipitate using antibodies specifically directed against the carboxyterminal part of the protein. Input corresponds to one tenth of the amount of the protein used in the assay. (B) Schematic representation of the Myrf isoform identified by [19] (upper bar, NCBI accession number Q3UR85.2), the splice variant used in this study (lower bar, NCBI accession number AAI57943.1) and various fragments used in interaction studies. Numbers represent amino acid positions. The DNA-binding Ntd80 domain is marked in grey. (C) Pulldown assays were performed with Sox10 fragments immobilized as GST-fusions on glutathione sepharose beads and the Myc-tagged Myrf fragments produced in HEK293 cell extracts. Detection of Myrf fragments was by western blot using an antibody directed against the Myc tag. Sox10 regions fused to GST included the dimerization and HMG domains (Dim/HMG), the K2 region and the transactivation domain (TA). (D–K) Transient transfections were performed in N2a cells with a luciferase reporter under control of the 727 bp Cx-47 1b promoter (D), the 416 bp Cx-32 P2 promoter (E), the 626 bp Mag promoter (F), the 1.2 kb WmN1 Plp enhancer (G), the 3 kb upstream region of the Mbp gene (H), a 631 bp conserved region 17 kb upstream of the Mbp gene (I), the 415 bp Mpz promoter (J) and the 1.3 kb MSE Krox20 enhancer (K). Empty pCMV5 expression plasmids (−) or expression plasmids for Sox10 and Myrf were co-transfected as indicated below the bars. Luciferase activities in extracts from transfected cells were determined in at least four experiments each performed in triplicates. The activity obtained for the luciferase reporter in the absence of ectopic transcription factor was arbitrarily set to 1. Fold inductions in the presence of transcription factors were calculated and are presented as mean ± SEM.