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

Figure 3.

Figure 3

Adgrv1 Y6236fsX1 mutation disrupts ALC formation. a,b) Whole‐mount immunostaining showing the localization of ALC components, a) WHRN and b) USH2A, in the stereocilia of IHC and OHC from P5 mice of different genotypes as indicated. TRITC‐phalloidin was used to visualize the F‐actin core of stereocilia. All images were taken from the middle turn of the basilar membrane using a confocal microscope. Scale bar: 2 µm. Representative images from three independent experiments were shown. c) Representative blotting showing coimmunoprecipitation of Flag‐ADGRV1 or Flag‐Y6236fsX1 with Myc‐WHRN and Myc‐USH2A in HEK293 cells. HEK293 cells were cotransfected with different combinations of ALC components as indicated in the figure. WT ADGRV1 or Y6236fsX1 mutant was immunoprecipitated by Flag antibody‐conjugated agarose, and the ALC components were detected by specific antibodies against Flag or Myc. d) Quantitative analysis of WHRN and USH2A levels that were coimmunoprecipitated with Flag‐ADGRV1. The data are correlated with Figure 3c and the band intensity of WHRN or USH2A co‐immunoprecipitated with Flag antibody‐conjugated agarose in the HEK293 cells transfected with Flag‐ADGRV1/Myc‐WHRN/Myc‐USH2A was used as the reference (normalized to 1). Data are from three independent experiments (n = 3). e) Representative blotting showing the ubiquitination levels of USH2A in HEK293 cells that were transfected with Myc‐WHRN, Flag‐USH2A, Myc‐ADGRV1 (WT or Y6236fsX1 mutant), and HA‐ubiquitin (HA‐UB) and were pretreated with lysosome inhibitor E64D. f) Quantitative analysis of the ubiquitination levels of Flag‐USH2A. The data are correlated with Figure 3e and the staining intensity of ubiquitinated USH2A (stain above 55 kd in the lane) in HEK293 cells transfected with Myc‐WHRN/Flag‐USH2A/HA‐Ub and treated with E64D was used as the reference (normalized to 1). Data are from three independent experiments (n = 3). Data information: d) ***p < 0.001; ns, no significant difference; HEK293 cells cotransfected with the other combinations of ALC components compared with those cotransfected with WT ADGRV1, USH2A, and WHRN. f) ***p < 0.001; HEK293 cells cotransfected with WHRN/USH2A/HA‐UB compared with those transfected with USH2A/HA‐UB. &&& p < 0.001; HEK293 cells transfected with ADGRV1/WHRN/USH2A/HA‐UB compared with those transfected with WHRN/USH2A/HA‐UB. ## p < 0.01; HEK293 cells transfected with Y6236fsX1 compared with those transfected with WT ADGRV1. The bars indicate the mean ± SEM values. All data were statistically analyzed using one‐way ANOVA with Dunnett's post‐hoc test.