Fig. 5: Functional analysis of the EDA·A1-EDAR interaction mutations.
a Immunofluorescence staining showing the binding of EDA·A1THD variants to EDAR on the cell membrane. MBP-EDA·A1THD, full-length EDAR-Flag and DNA were stained in green, red and blue, respectively. Scale bar: 10 µm. b Luciferase reporter assay shows that supplemented WT EDA·A1THD activates NF-κB signaling pathway in a dose-dependent manner in HEK293T cells ectopically expressing full-length EDAR. Data are presented as the mean ± SEM for n = 3 independent experiments. c The interaction between EDARCRDS and EDA·A1THD variants was assessed by a luciferase reporter assay with HEK293T cells ectopically expressing full-length EDAR (n = 3, mean ± SEM). Top right: similar amount of WT and mutant soluble EDA·A1THD proteins from supernatants of transfected HEK293T cells were analyzed by Western blot to validate the amounts of proteins. d Luciferase reporter assay with HaCaT cells that express endogenous EDAR (n = 3, mean ± SEM). A two-sided Student t-test was performed. ***P = 0.00016 (A259E), ***P = 0.000043 (D265G), **P = 0.0045 (R276C) and ***P = 0.000033 (Mock). Source data are provided as a Source Data file.
