Figure 6.

Coexpression of UPIa/UPII and UPIb/UPIII pairs results in their colocalization at the plasma membrane. Cotransfection of 293T cells was carried out as described in Figures 3 and 4. Cells were fixed and incubated with the first antibodies against the UP subunits and the HA or myc tag, followed by incubation with the secondary antibodies used in Figure 2A. Immunostaining was performed on nonpermeabilized cells that were analyzed by confocal microscopy. When UPIa (b) and HA-UPII (a) were coexpressed, both subunits colocalized at the plasma membrane. This was not the case when UPIa was coexpressed with myc-UPIII, but ER staining was observed in permeabilized cells (our unpublished data). On the other hand, when UPIb (e) was coexpressed with myc-UPIII (d), both UP subunits were expressed at the plasma membrane (f), but HA-UPII cannot be detected at the plasma membrane of cells expressing UPIb and HA-UPII (g). As may be expected from our single-transfection experiments, UPIb reached the cell surface (h) when coexpressed with HA-UPII.