Fig. 3. Colocalization of GFP-Rab11 wt or mutants with rhodamine-transferrin. Cells were incubated in RPMI (serum free) containing 20 µg/ml human Tf (tetramethyl rhodamine-conjugated) for 45 minutes at 37°C and washed twice with ice-cold PBS. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. Images were obtained with a CCD camera (Orca I, Hamamatsu) and processed using MetaMorph 4.5 (Universal Images Corporation). Left panels: GFP-Rab11wt and mutants. Middle panels: Rhodamine-transferrin (Rh-Tf). Right panels: overlay. In the first three panels, the focus was set on the small Rab11 structures that colocalize with the internalized Rh-Tf. In contrast, in the bottom panels the focus was set on the large ring-shaped structures formed in cells transfected with the mutant Q70L. Rh-Tf was not detected in these structures.