Fig. 6. Effect of CFP-Rab5-CAAX on EGF receptor endocytosis. CFP-Rab5-CAAX, CFP-Rab5(Q79L)-CAAX and CFPRab5(S34N)-CAAX were transiently cotransfected with ß2-YFP in PAE/EGFR cells. The cells were incubated with 1 ng/ml EGF-Rh for 10 minutes at 37°C and fixed. EGF-Rh, YFP and CFP were detected using Cy3, YFP and CFP filter channels. Optical sections close to the bottom of the cell are shown. 'Yellow' signifies colocalization of YFP and rhodamine fluorescence. 'Cyan' signifies colocalization of CFP and rhodamine fluorescence. Insets show an enlargement of the outlined regions. Additional insets (lower panel, ß2-YFP image) show the merge images of EGF-Rh and ß2-YFP, in which the EGF-Rh image (red) was right-shifted 4 pixels (250 nm) to allow better visualization of colocalization of red and green dots. The arrows point to a cell that does not express CFP-Rab5(S34N)CAAX and that displays typical endosomal localization of EGF-Rh. Note colocalization of EGF-Rh with CFP-Rab5(Q79L)-CAAX and CFP-Rab5-CAAX in endosomes is seen as 'donut-shape' structures. Bar, 10 µM.