Fig. 6. Colocalization of ß₁AR-GFP with transferrin and with clathrin. (A-I) HEK cells transiently transfected with ß₁AR-GFP without (D-F) or with (A-C,G-I) arrestin-2 were incubated at 4°C with Texas Red®-labeled transferrin (20 µg/ml) for 30 minutes and with 1 µM ISO at 37°C for another 20 minutes (D-I). The cells were washed and fixed as described in Materials and Methods. The distribution of ß₁AR-GFP (green) and transferrin (red) was examined using a confocal microscope. Colocalization of transferrin with ß₁AR-GFP (yellow) can be observed in the merged images. (J-R) Same as A-I except transferrin was omitted and the cells were permeabilized and stained for clathrin (red) with rabbit anti-clathrin followed by Cy5-conjugated anti-rabbit (color changed from blue). Colocalization of clathrin with ß₁AR-GFP (yellow) can be observed in the merged images. Scale bar: 5 µm.