Fig. 3

Fig. 3 Cc2d2a localizes to the connecting cilium/transition zone of cilia in photoreceptors and other cell types. A–C′′′) Retinal cryosections from cc2d2a+/+ embryos labeled with anti-Cc2d2a (green) and anti-acetylated tubulin [red in (A–A′′′)], anti-Ift88 [red in (B–B′′′)] and anti-gamma-tubulin [red in (C–C′′′)] show Cc2d2a localization at the base of cilia (arrows). The Cc2d2a signal is apical to the basal-most acetylated tubulin signal [arrowhead in (A′′)], apical to the gamma-tubulin signal (C–C′′′) and within the Ift88-poor region that represents the connecting cilium, between the brightest Ift88 signal and a fainter, more basal signal [arrowheads in (B′ and B′′)]. Embryos are 4 d.p.f. in (A) and 5 d.p.f. in (B and C). (D–G) Immunofluorescence against Cc2d2a (green) and Arl13b (red) showing Cc2d2a localization at the base of 48 h.p.f. pronephric duct cilia (D and D′), 24 h.p.f. floorplate cilia (E and E′), 72 h.p.f. olfactory pit cilia (F) and 24 h.p.f. cerebellar neuronal progenitor cilia (G). (H) High power confocal image of a neuronal progenitor cilium from a 24 h.p.f. arl13b-GFP embryo stained with antibodies against gamma-tubulin (green), Cc2d2a (red) and GFP (blue). (D–H) Single-confocal sections of whole-mount embryos. Scale bars are 6 µm in (A), 2 µm in (A′), 0.4 µm in (A′′ and A′′′), 4 µm in (B and C), 1 µm in (B′–B′′′), 0.5 µm in (C′–C′′′), 5 µm in (D and F), 10 µm in (E), 20 µm in (G) (2 µm in inset) and 0.6 µm in (H). (I) Western blot with Cc2d2a P2D9 antibody from whole wild-type, zygotic and maternal zygotic (mz) cc2d2a-/- embryo lysates at 48 h.p.f. The Cc2d2a band appears as a doublet around 190 kDa in wild-type (cc2d2a+/+) and is absent in zygotic and mz cc2d2a-/- embryos. (J) Western blot with Cc2d2a P2D9 antibody on full-length and truncated (W628*) Cc2d2a-GFP protein overexpressed in HEK293T cells. The antibody recognizes the full-length as well as the truncated Cc2d2a protein.