Figure 3.

(A) Staining of α₂δ₄ subunit in WT and mutant (MUT) animals. The arrow in the WT images shows the punctate labeling for α₂δ₄ subunit in the outer plexiform layer (OPL). Puncta are absent in mutant retina. (B) Co-localization of the α₁ subunit of calcium channels (Pan-α₁, green) and photoreceptor ribbon (PSD95, red) in WT retina. In mutant retina the structure of presynaptic terminal is disorganized as shown by the lack of horseshoe-shaped PSD95 staining. Bottom images show magnification of the boxed area. (C) Western blot analysis of TMEM16A content in retinas of WT and MUT animals. Left images: Example western blots. Right histograms: Optical density ratio TMEM16A/β-actin in WT and MUT mice. Values expressed as average ± SEM, n = 8 mice per condition, p = 0.94, t-test. (D) Immunoblot in retinal tissue from WT and MUT mice with antibodies against the α₁ subunit (top row) and the α₂δ₄ subunit (middle row) of calcium channels, either in whole lysate (left) or after co-immunoprecipitation of TMEM16A (right). The bottom row shows the control labeling against TMEM16A (input). Representative example of N = 3 independent Co-IP experiments. Each experiment is the measure obtained from retinas of n = 4 animals. (E) Immunoblot in retinal tissue from WT and MUT mice with the antibody against Cav1.2 subunit of calcium channel (top row) either in whole lysate (left lanes), after co-immunoprecipitation of TMEM16A (middle lanes) or in control supernatant from co-immunoprecipitation (right lanes). The control labeling against TMEM16A (input) is shown in the bottom panels. Representative example of N = 3 independent Co-IP experiments. Each experiment is the measure obtained from retinas of n = 4 animals.