Fig. 6. Subcellular distribution and cell-surface targeting of HA-GLUT8 in AP-2-depleted HeLa cells. (A) Cells were grown on coverslips and co-transfected with HA-GLUT8 and empty pSuper vector, HA-GLUT8 and pSuper/µ2-1, and HA-GLUT8-LL/AA and empty pSuper vector. After 6 days in culture, non-permeabilized cells were stained for the HA-epitope tag and analyzed by confocal laser scanning microscopy as described in the Materials and Methods. Bars, 20 µm. (B) Cells were co-transfected with HA-GLUT8s and empty pSuper vector (-) or pSuper/µ2-1 construct (+), and cultured for 6 days. Then, the cell-surface levels of the HA-GLUTs were determined using an antibody binding assay as described in the Materials and Methods. In parallel, the expression of the HA-GLUTs was determined by western blot using an anti-HA antibody (inset). The cell surface-associated radioactivity was normalized to the level of the HA-GLUT8-LL/AA mutant. Results are the means ± s.e.m. of four to six independent experiments performed in duplicate. ^*P<0.05 (paired Student's t-test) comparing pSuper/µ2-1 cells and control cells.