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Fig. 5
Itga8 and Pcdh15a interact in hair cells. (A–D) Representative immunoblots of co-IP studies from 3 dpf larvae showing Itga8–Pcdhs15 interaction. IP itga8: Itga8 IP; IP NGpS: IP with pre-immune guinea pig serum; IP 15a, Pcdh15a IP; IP NRS, IP with pre-immune rabbit serum; IB 15a, Pcdh15a immunoblot; IB Itga8, Itga8 immunoblot. Asterisks denote specific bands (red asterisks, full-length protein; black asterisks, small variants). Three independent experiments were performed. (E–H) Coronal sections of 5 dpf WT (E,H) orbiter (orb) strong (F) and weak (G) mutants. Positive PLA (red dots) (E and high magnification inset), demonstrate an in situ Itga8–Pcdh15a association in WT but not in pcdh15a mutants (F,G). (H) Negative control in which one of the primary antibodies (in this case anti-Itga8) was omitted. Sections were immunostained for acetylated tubulin (blue) and counterstained with phalloidin (green). (I) Cartoon of a neuromast (lateral view) showing the stained structures: hair bundle in green, kinocilia and cell bodies in blue and the Itga8–Pcdh15a complex as red dots. Ten animals from two independent experiments were inspected. Scale bar: 6 µm (for E–H), 10 µm (inset in E).