Fig. 4. Effect of Rab5 mutant overexpression on EGF receptor internalization. (A) Wild-type CFP-Rab5 (wt) and CFP-Rab5(S34N) mutant were transiently co-expressed 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 from the bottom of the cells 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. An additional inset (lower panel, ß2-YFP image) shows the merge image of EGF-Rh and ß2-YFP, in which the EGF-Rh image (red) was left-shifted 4 pixels (250 nm) to allow better visualization of colocalization of red and green dots. The arrow points to a cell that does not express CFP-Rab5(S34N) and displays typical endosomal localization of EGF-Rh. The images are representative of at least four independent experiments. Bar, 10 µm. (B) GFP-dynamin mutant (DynK44A) was transiently expressed in PAE/EGFR cells. Cells were incubated with 1 ng/ml EGF-Rh for 10 minutes at 37°C. EGF-Rh and GFP-dynamin were detected using Cy3 and GFP filter channels. Insets show an enlargement of the outlined regions. 'Yellow' signifies colocalization of GFP and rhodamine fluorescence. The images are representative of at least four independent experiments. Bar, 10 µm.