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. 2023 Apr 17;10(16):2205993. doi: 10.1002/advs.202205993

Figure 6.

Figure 6

Potential interaction mode of the ALC revealed by ITC and FlAsH‐BRET. a) Interactions between the first two PDZ domains of WHRN and synthetic USH2A/ADGRV1 C‐terminal peptides revealed by isothermal titration calorimetry (ITC). The 10‐residue C‐terminal peptides of both ADGRV1 and USH2A harboring the PDZ‐binding motif (denoted as ADGRV1p and USH2Ap) were synthesized and titrated with purified WHRN‐PDZ1 and WHRN‐PDZ2 proteins. b) Representative blotting and c) quantitative analysis of the USH2A or ADGRV1 levels coimmunoprecipitated with the WT or PDZ domain‐truncated WHRN. Data are from three independent experiments (n = 3). d) Schematics illustrating the ADGRV1 with a FlAsH motif (CCPGCC) incorporated at corresponding sites in ICL1, ICL2, or ICL3 and the WHRN with a NanoLuc (Nluc) moiety inserted at distinct sites flanking the three PDZ domains (denoted as L‐Z1, Z1‐L, L‐Z2, Z2‐L, L‐Z3, and Z3‐L, respectively). e) Saturation BRET signal between NLuc and FlAsH in HEK293 cells co‐transfected with a fixed amount of NLuc‐WHRN plasmid and an increasing amount of ADGRV1‐FlAsH plasmids. Data are from three independent experiments (n = 3). Data information: c) ***p < 0.001; ns: no significant difference; HEK293 cells transfected with WHRN mutants compared with those transfected with WT WHRN. The bars indicate the mean ± SEM values. All data were statistically analyzed using one‐way ANOVA with Dunnett's post‐hoc test.