Fig. 5. Transport of CsA-Rh50 from the plasma membrane to the CV. Cells expressing pFL759 were incubated with the anti-CsA antibody for 5 minutes at 4°C, washed and then warmed up for the times indicated on the left. The amount of CsA-Rh50 left on the cell surface after internalization was monitored by incubating cells at 4°C for 30 minutes with an anti-mouse secondary antibody coupled to Alexa Fluor 647 (CsA Surface, blue staining). Then, cells were fixed, methanol-permeabilized and incubated with an anti-mouse secondary antibody coupled to Alexa Fluor 568 to detect internalized CsA-Rh50 as well as any remaining construct on the cell surface (CsA Total, red staining). The CV compartment was decorated with a polyclonal antibody to Rh50 (RH153, green staining). Cells were observed by confocal microscopy. CsA-Rh50 was rapidly internalized, accumulated in vesicular structures and after 60 minutes of internalization localized on the CV network. Bar, 10 µm.