Fig. 6. Internalized CsA-Rh50 is excluded from p80-containing endosomes and from the Golgi apparatus. (A) Cells expressing pFL759 were treated as described in Fig. 5. At the end of the procedure, early and late endosomal vacuoles were labeled with the monoclonal antibody to p80 (H161) directly coupled to Alexa Fluor 488 (p80, green staining). CsA-Rh50 barely colocalized with p80 endosomes even after 2 hours of internalization. Note that the CV compartment was not detected here upon staining with the anti-CsA antibody because p80 endosomes and the CV localize in different areas of the cell and that we selected by confocal microscopy a plane appropriate for the observation of p80 endosomes but excluding the CV network. (B) The Golgi marker golvesin (C)-GFP was expressed in cells expressing pFL759. Cells were treated as described in Fig. 5. After 5 minutes of internalization, CsA-Rh50 did not colocalize in golvesin (C)-GFP positive structures. Bar, 10 µm.