Figure 2

Sox10 is necessary and sufficient for the activity of ERBB3 _MCS6 in vitro. (A) Luciferase activities of wild-type ERBB3_MCS6 and TFBS mutations in ERBB3_MCS6 in melan-A cell line. The position of each TFBS in ERBB3_MCS6 is shown on top and the corresponding mutation in each TFBS is shown next to the luciferase value for that construct. * indicates statistical significance. (B) Luciferase activity of WT ERBB3_MCS6 in melan-A cells in mock-transfected cells and in cells with transient Sox10 and Ap2 knockdown. (C) Western blot to confirm knockdown of Sox10 and Ap2 protein upon siRNA treatment. Tubulin antibody was used as a loading control. (D) Western blot showing Erbb3 protein levels in melan-a cells upon transient transfection with Sox10 and Ap2 siRNA or mock-transfected cells. Tubulin antibody was used as a loading control. (E) Luciferase activity of ERBB3_MCS6 upon knockdown of Sox10 in S16 cells using a dominant negative SOX10 mutant (E189X) under a CMV prmoter. (F) Luciferase assay of WT and SOXE-2m ERBB3_MCS6 in Neuro2A cells when transiently co-transfected with an empty expression vector (pcDNA.31) and Sox10 cDNA. Cell lysates were collected 24 hours post transfection. (G) Luciferase assay of WT ERBB3_MCS6 in Neuro2A cells when transiently co-transfected with equal amounts of WT and Sox10-ΔSTP cDNA either individually or in combination. Cell lysates were collected 24 hours post transfection. All luciferase values are normalized to renilla internal control and shown as fold-change compared to promoter only construct (pe1B) with standard deviation. (H) Real-time PCR of Erbb3 transcript levels upon expression of WT and Sox10-ΔSTP cDNA individually and in combination. Values are normalized to 18s internal control and are shown as fold-change compared to promoter only construct (pcDNA3.1) with standard error. (I) Real-time PCR of ChIP against Sox10 in untreated S16 cells and in S16 cells treated with Sox10 morpholino. Black bar indicates enrichment upon Sox10 ChIP whereas grey bar indicates enrichment with non-specific IgG. Error bars indicate standard deviation of technical replicates in real-time PCR.