Figure 3
From: MITF variants cause nonsyndromic sensorineural hearing loss with autosomal recessive inheritance
Reporter assay and western blot analysis (a) The basal luciferase level of the empty vector was 32. Overexpression of wild-type MITF induced TYR promoter activity by up to 9.4-fold (300/32), R341C at 5.1-fold (164/32 or 54%), and R341H at 4.3-fold (139/32 or 46%), respectively, (p < 0.0001 by one-way ANOVA). (b) Western blot analysis. Left panel: upper image demonstrating the blot of MITF, from left to right, molecular size marker; wildtype MITF-isoform A, R341C, and R341H variants (labeled as R341H-1), the wild-type, R341C and R341H protein with normal predicted size. Lower image of the left panel showing the blot of GAPDH used as a loading control for normalization for wildtype MITF and each variant. Noted almost equivalent amount of the GAPDH in all lanes. Detail of GAPDH staining was described in the Supplementary Figure S1. Brightness and contrast of the upper and lower images were not modified from its original appearances The right panel denoting a greater amount of the R341C protein and a decreased quantity of the R341H protein expressed, compared with the wild type [p-value 0.0011 and 0.0898, respectively, using ANOVA with post hoc (Tukey) test]. The relative level of band intensity of wild-type MITF was set as 1. (c) Diagram showing the Luc-reporter constructs, with putative MITF binding sites shown as a filled box (Tyrosinase distal enhancer or TDE, M-box, E-box).
