Fig. 8. The effect of CFP-Rab5-CAAX proteins on transferrin endocytosis. (A) CFP-Rab5-CAAX or CFPRab5(S34N)-CAAX were transiently co-expressed with ß2-YFP in PAE/EGFR cells. The cells were incubated with 5 µg/ml Trf-TR for 10 minutes at 37°C and fixed. Tfr-TR, YFP and CFP were detected using Cy3, YFP and CFP filter channels. 'Yellow' signifies colocalization of YFP and rhodamine fluorescence, whereas 'cyan' signifies colocalization of CFP and rhodamine fluorescence. Insets show an enlargement of the outlined regions. Additional inset (low panel, YFP image) shows the merge image of Trf-TR and ß2-YFP, in which the Trf-TR image (red) was right-shifted 4 pixels (250 nm) to allow better visualization of the overlap of red and green dots. The rhodamine image (low panel) is shown overexposed to visualize low levels of Trf-TR fluorescence in the cell expressing CFPRab5(S34N)-CAAX. This resulted in saturation of intense endosomal signals of Trf-TR in neighboring cells not expressing CFP-Rab5(S34N)-CAAX. Bar, 10 µM. (B) Gallery of high-magnification images from several cells expressing CFP-Rab5(S34N)-CAAX and treated as in (A) that show Tfr-TR colocalization with ß2-YFP.