Fig. 4: Biochemical analysis of mutations at the EDA·A1THD-EDARCRDS interface.
a Pull-down assay using anti-Flag beads with ectopically expressed human Flag-EDA·A1THD and purified EDARCRDS-MBP. The levels of each protein in the input and pull-down samples were analyzed by immunoblotting with the indicated antibodies. The upper and lower bands of Flag-EDA·A1THD are N-glycosylated and non-N-glycosylated isoforms, respectively. WT, wild type; MBP (EDAR), anti-MBP to detect EDARCRDS-MBP; Flag (EDA), anti-Flag to detect Flag-EDA·A1THD. b SDS-PAGE analysis of EDA·A1THD variants and EDARCRDS. c Surface plasmon resonance measurement of binding affinity between EDA·A1THD variants and EDARCRDS. d Deglycosylation of EDA·A1THD. Soluble Flag-EDA·A1THD proteins expressed in the supernatant of HEK293T cells were treated with or without peptide N-glycanase (PNGase) F for 12 hours (12 h) or 24 hours (24 h) before western blotting analysis. N-glycosylated forms of Flag-EDA·A1THD (upper bands) disappeared after being treated with PNGase F. e Pull-down assay using amylose (MBP-tag affinity) agarose beads with ectopically expressed human Flag-EDA·A1THD and purified EDARCRDS-MBP. The levels of each protein in the input and pull-down samples were analyzed by immunoblotting with the indicated antibodies. WT, wild type; MBP (EDAR), anti-MBP to detect EDARCRDS-MBP; Flag (EDA), anti-Flag to detect Flag-EDA·A1THD. f Pull-down assay using anti-Flag beads with ectopically expressed mouse Flag-EDA·A1THD and purified mouse EDARCRDS-MBP. The levels of each protein in the input and pull-down samples were analyzed by immunoblotting with the indicated antibodies. Source data are provided as a Source Data file.
