Figure 6. Mutation of STAT6-binding elements within the Il31 promoter impairs promoter activity.

A. Schematic representation of reporter constructs from the human Il31 promoter. A 474-bp fragment of the human Il31 promoter was cloned into the pGL3 Basic luciferase reporter plasmid. Mutations were introduced by site-directed mutagenesis into the NF-κB-binding site or into one or both STAT6-binding elements. B. Reporter gene assay of HEKblue IL-4/IL-13 cells transfected with the wild-type (WT) Il31 promoter construct and either an expression vector encoding the IL-33 receptor subunit ST2L or empty pEF-Bos vector. The day after the transfection, cells were induced with 50 ng/ml IL-4 and/or 30 ng/ml IL-33. After 24 hours of stimulation, luciferase activity was assessed. Data represent mean values of three independent experiments carried out in duplicates; error bars indicate standard deviations. ALU arbitrary light units, ui uninduced. C. Reporter gene assay of HEKblue IL-4/IL-13 cells transfected with the wild-type (WT) Il31 promoter construct or mutated versions thereof. The expression vector encoding human ST2L was co-transfected. Data were normalized to the corresponding uninduced sample. Mean values of two independent experiments carried out in duplicates are shown. Error bars indicate standard deviations. Statistical analyses: one-way ANOVA/Tukey’s multiple comparisons test. **** p ≤ 0.0001.