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

Figure 5.

Figure 5

Phosphorylated WHRN regulates USH2A ubiquitination through recruiting WDSUB1. a) Representative blotting and b) quantitative analysis of the coimmunoprecipitation of WHRN with WDSUB1 in the lysates of cochlea isolated from WT or Y6236fsX1 mutant mice. Data are from three independent experiments (n = 3). c) Representative blotting and d) quantitative analysis of USH2A ubiquitination in HEK293 cells transfected with Flag‐USH2A and HA‐UB in the absence or presence of GFP‐WDSUB1 and Myc‐WHRN. Data are from three independent experiments (n = 3). e) Representative blotting and f) quantitative analysis of USH2A ubiquitination in HEK293 cells transfected with Flag‐USH2A, Myc‐WHRN, and HA‐UB in the presence of control or WDSUB1 siRNA. Data are from three independent experiments (n = 3). g) Quantitative analysis of the GFP‐WSDUB1 coimmunoprecipitated with Flag‐WHRN (WT or phospho‐mimic mutants) in HEK293 cells. Data are correlated with Figure S6h, Supporting Information and are from three independent experiments (n = 3). h) The stability of USH2A in ADGRV1 (WT or Y6236fsX1) overexpressed HEK293 cells that were treated with control or WDSUB1 siRNA. Data are correlated with Figure S6i, Supporting Information and are from three independent experiments (n = 3). Data information: b) ***p < 0.001; ns: no significant difference; WDSUB1 immunoprecipitated with WHRN compared with those immunoprecipitated with IgG. ### p < 0.001; WDSUB1 immunoprecipitated in Y6236fsX1 mutant mice compared with that in WT mice. d) **p < 0.01; HEK293 cells transfected with USH2A/UB/WDSUB1 compared with those transfected with USH2A/UB. ### p < 0.001; HEK293 cells transfected with USH2A/UB/WDSUB1/WHRN compared with those transfected with USH2A/UB/WDSUB1. f) ***p < 0.001; ns: no significant difference; HEK293 cells transfected with USH2A/WHRN/UB compared with those treated with the same siRNA but transfected only with USH2A/UB. ### p < 0.001; HEK293 cells treated with siWDSUB1 compared with those treated with control siRNA. g) ***p < 0.001; ns, no significant difference; HEK293 cells transfected with WHRN mutants compared with those transfected with WT WHRN. h) *p < 0.05; **p < 0.01; HEK293 cells treated with siWDSUB1 compared with those treated with control siRNA. ns: no significant difference; HEK293 cells transfected with Y6236fsX1 mutant compared with those transfected with WT ADGRV1. The bars indicate the mean ± SEM values. All data were statistically analyzed using b,d,f,g) one‐way or h) two‐way ANOVA with Dunnett's post‐hoc test.