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. 2011 Sep 9;12(10):1055–1061. doi: 10.1038/embor.2011.175

Figure 3.

Figure 3

Clathrin endocytosis is required for Rspo3 signalling. (A–L) Clathrin is colocalized with Rspo3 and is required for Rspo3 internalization. Confocal microscopy of HepG2 cells treated for 1 h with horseradish peroxidase (HRP)-tagged Rspo3-ΔC on ice (A), or at 37°C (B,D–L), or in the presence of Flag-tagged Rspo3-ΔC (C). HRP-tagged Rspo3-ΔC was visualized by tyramide signal amplification without or with coimmunofluorescence of anti-green fluorescent protein (GFP) antibody against overexpressed xtLGR4-EYFP (D), Clathrin–GFP (E) and Caveolin–GFP (F). Where indicated, cells were pretreated with small interfering RNAs (siRNAs) for 4 days (B,G–I) or with endocytic inhibitors for 1 h (J–L). (M) Clathrin is required for Rspo3-induced nuclear β-catenin accumulation. Confocal microscopy of NTERA2 cells incubated for 1 h with endocytic inhibitors or pretreated with the indicated siRNAs for 3 days. Cells were then treated for 4 h with Wnt3a, or Wnt3a together with Rspo3, or 50 mM LiCl. Scale bars indicate 10 μm. MDC, monodansylcadaverine.