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. 2013 Mar 1;24(5):555–565. doi: 10.1091/mbc.E12-06-0437

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© 2013 Zilber et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Fuzzy regulates generation of cilia and ciliary length. (A) Left, mouse embryonic fibroblasts (MEFs^Fuz/Fuz) derived from Fuzzy^−/− embryos are unciliated; middle, expression of Fuzzy^Flag in MEFs^Fuz/Fuz restores cilia visualized with anti–acetylated-α-tubulin antibody (red, arrows, and inset); right, primary cilium (red, arrow, inset) can be visualized only in the MEFs^Fuz/Fuz cell transfected with Fuzzy^Flag cDNA (identified with anti-FLAG antibody, green and inset); bar, 10 μm. (B) In unciliated MEFs^+/+, endogenous Fuzzy (red, visualized with specific rabbit anti-Fuzzy antibody) is localized to the cytoplasmic vesicles; bar, 2 μm. (C) Endogenous Fuzzy protein is undetectable in MEFs^Fuz/Fuz; bar 2 μm. (D) Negative control: MEFs^+/+ stained with only secondary antibody; bar, 2 μm. (E) In ciliated MEFs^+/+, endogenous Fuzzy (red, arrowhead) is enriched at the basal body (green) detected with anti–γ-tubulin antibody; only Z-stacks at the basal body level were merged; bar, 2 μm. (F) In ciliated MEFs^+/+, endogenous Fuzzy protein (red) is localized to the primary cilium (arrows, visualized with anti–acetylated-α-tubulin antibody, green staining), only Z-stacks that contain ciliary images were merged; bar, 2 μm. (G) Fuzzy^GFP is localized to ciliary axoneme in MEFs^Fuz/Fuz transfected with Fuzzy^GFP cDNA. (H) MDCK cells stably overexpressing Fuzzy^GFP produce long cilia, and Fuzzy^GFP is detected at the basal body (arrowhead) and ciliary axoneme (arrows); both basal body and ciliary axoneme are visualized with anti–acetylated- α-tubulin antibody in cells permeabilized with 0.4% Triton; bar, 2 μm. (I) In nonconfluent unciliated MDCK cells, Fuzzy^GFP is localized to the cytosplasmic vesicles (arrows); bar, 10 μm.