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Fig. 4. Co-staining of HA-Dvl-2-EGFP with endocytic markers. (A-F) HEK293 cells immunostained with antibodies against endogenous EEA-1 (A,B), transferrin receptor (C,D) and CD63 (E,F). Antibody localisation is shown in red. There was no non-specific background staining when control isotype-specific antibodies was used as the primary antibody (data not shown). Nuclei were counterstained with ToPro III (blue). A,C,E, non-transfected cells. B,D,F, cells transfected with HA-Dvl-2-GFP. (G-H) Localisation of Alexa-594-coupled transferrin in HA-Dvl-2-GFP-transfected CHO cells, identifying peripheral transferrin-containing vesicles and early endosomes (G) or perinuclear recycling endosomes (H). (I) Localisation of late endosomes in HA-Dvl-2-GFP transfected CHO cells using an antibody to LgpB. Note that HA-Dvl-2-GFP failed to colocalise with any of these markers. (J) Analysis of HA-Dvl-2-GFP puncta using electron microscopy of HA-Dvl-2-GFP transfected HEK293 cells. The nuclear membrane (arrowheads) is clearly seen around the nucleus (n). In contrast, there is no evidence of a membrane around the electron-dense body (p), in which the HA-Dvl-2-GFP is concentrated, as shown by the anti-HA immuno-gold labelling in the magnified inset box (arrows). Bars, 5 µm (A-F); 20 µm (G-I); 1 µm (J).

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