Fig. 3. SKD1(E235Q)-expression inhibits the surface appearance of endolyn. NRK cells expressing GFP-SKD1(E235Q) were fixed and stained with mAb against endolyn and then with Alexa594-conjugated secondary antibodies (A,B). Panel A shows the merged image of GFP-SKD1(E235Q) (green) and endolyn (red). Note that the infected cells (asterisks) exhibited enlarged structures (discussed in Fig. 6,7). The cell-surface endolyn was labeled with mAb at 4°C, then fixed immediately (C,D). Note that the surface expression of endolyn is significantly reduced in cells expressing SKD1(E235Q) (asterisks in C,D). To monitor the fate of the internalized mAb, surface-labeled cells were washed and incubated at 37°C for 1 hour before fixation and then stained with Alexa594-conjugated secondary antibodies (E,F). Note that the amount of internalized mAb in infected cells (asterisk in E and F) is less than that in uninfected cells. They are accumulated into the E235Q compartments in infected cells while they reach the lysosomes in uninfected cells (E,F). Panel C and E shows the merged image of GFP-SKD1(E235Q) (green) and the location of internalized mAb (red). The cell surface binding and the subsequent internalization of ¹²⁵I-mAb to endolyn were reduced in the cells expressing SKD1(E235Q) (G,H), approximately 50 and 70% of controls, respectively. The amount of ¹²⁵I-mAb associated to cell surface at 4°C (G) and internalized during the indicated times at 37°C (H) were represented as the cpm/µg cell protein. Data were obtained from two (G) or three (H) independent experiments, respectively.