Figure 2
From: Adenylyl cyclase 6 plays a minor role in the mouse inner ear and retina
Gα and PKA localization in cochlear stereocilia and PKA activity reduction in Adcy6−/− cochleas. (a) The specificity of Gαs and Gαi antibodies was verified by immunoblot analysis using their corresponding FLAG-tagged recombinant proteins transfected in HEK293 cells. The immunoblot detected by a FLAG antibody (right) was used to confirm the transfected Gα protein expressions. NC: untransfected HEK293 cell lysate. The original uncropped blot images are presented in Supplementary Fig. S4. (b) Immunostaining shows that Gαs protein (magenta) is located along the entire stereocilia (green) except the tip (arrows) and base (filled arrow) in a wild-type cochlear hair cell at P4. (c) Immunostaining shows that Gαi protein (magenta) is located at the very tip (arrow) and around the ankle link complex region (filled arrow) in wild-type cochlear stereocilia at P4. (d) Immunostaining shows that pan-PKA Cα1 (PKA, magenta, upper row) and phospho-PKA Cα1 (pPKA, magenta, middle row) are located at the basal portion of stereocilia in wild-type IHCs (left) and OHCs (right). The distribution of phospho-PKA Cα1 subunit in Adcy6−/− cochlear hair cells (lower row) is similar to that in wild-type cochlear hair cells (middle row). (b–d) The merged images correspond to the single-channel magenta images. Scale bars: 2 µm. (e) Immunoblot analysis shows that pan-PKA Cα1 expression levels in Adcy6−/− (−/−) cochleas and vestibular organs are similar to those in wild-type (WT) cochleas and vestibular organs. Phospho-PKA Cα1 subunit level is reduced by ~ 50% in Adcy6−/− cochleas but remains normal in Adcy6−/− vestibular organs. The blot images are representative images from three to four biologically independent experiments. γ-tubulin was used as a loading control. The original uncropped blot images are presented in Supplementary Fig. S4. (f) Quantification of the phospho-PKA Cα1 immunoblot signal intensities from three to four biologically independent experiments. The data are presented as ratios of phospho-PKA Cα1 to pan-PKA Cα1 and are normalized by corresponding wild-type ratios on the same blots. *, P value = 0.029 (Mann–Whitney test). Each point represents an individual biological sample pooled from at least 4 pups. Mean and SEM (standard error of the mean) are shown.
