Subtilisin treatment disrupts ankle links but not the ALC in cochlear stereociliary bundles. Subtilisin treatment eliminates ankle links in P4 cochlear hair cells, shown by a lack of immunoreactivity with an antibody directed against the GPR98 extracellular region (A). However, this treatment does not affect distribution of the GPR98 intracellular fragment at the P4 stereociliary base, shown by immunofluorescence using an antibody against the GPR98 cytoplasmic region (B). Localization of usherin (C), PDZD7 (D) and whirlin (E) at P4 stereociliary bases appears normal after subtilisin treatment (right column) compared with PBS-treated negative controls (left column). Stereociliary bundles are viewed from the front and side except the PBS-treated IHC bundle in (B), which is a back view. Arrows point to stereociliary bases. Green signals are from phalloidin staining. Magenta signals in stereociliary bundles are specific signals of USH2 and PDZD7 proteins, while magenta signals outside stereociliary bundles are non-specific. Scale bars, 1 µm.