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. 2009 Nov 2;187(3):365–374. doi: 10.1083/jcb.200907126

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© 2009 Milenkovic et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — Smo present on the cell surface translocates to the primary cilium after Shh stimulation. (A) Extracellular domains of Smo are recognized by anti-YFP (YFP tag), anti-SmoN (cysteine-rich domain), or the SNAP substrate (SNAP tag). An intracellular region of Smo is recognized by anti-SmoC. (B) Cell surface proteins were biotinylated, isolated on streptavidin beads, and examined for the presence of Smo or a control intracellular protein (p38) by immunoblotting. (C–E) Live YFP-Smo cells (Rohatgi et al., 2009) were exposed to anti-YFP (C and D) or anti-SmoN (E) according to the timeline shown to the left of each panel. (C) Insets (enlarged views of the boxed regions) show cilia visualized as shifted overlays of the color channels. (D) Intensity of Smo fluorescence at cilia, shown as fold increase, after treatment with Shh in cells pretreated with anti-YFP or anti-SmoC (control). Data indicate mean ± SEM. (E) Shh was added after cells were treated with anti-SmoN. Both the main panels and insets (enlarged views of the boxed regions) showing cilia are shifted overlays. Bars, 5 µm.