Transfected NSP4 travels to the cell surface in the absence of other viral proteins. A. BHK-21 cells were transfected with pcDNA3.2 NSP41-175 and surface biotinylated at 20 h post transfection at 4°C. Cells were lysed and surface proteins were precipitated with streptavidin agarose (left panel). Mock transfected cells were treated identically and served as controls (middle panel). Lanes 1 are the cell lysates from transfected or un-transfected cells and lanes 2 are the surface biotinylated, streptavidin pull downs of the transfected (left) or mock-transfected (center) cells. Lanes 3 show the transfected or mock-transfected lysates following biotinylation and streptavidin pull-down. RV-infected cell lysate (right panel) is shown as a NSP4 control. Note that substrate with femtogram sensitivity was used to detect the expression of transfected NSP4. B. BHK-21 cells were grown on glass cover slips, transfected with pcDNA3.2 NSP41-175, surface biotinylated in the cold, and probed with affinity-purified anti-NSP4150-175 and anti-rabbit IgG-CY2 (all panels) and either streptavidin-CY5 (top panels) or mouse anti- Na+/K+-ATPase and anti-mouse IgA-Texas Red (bottom panels,). Cells were visualized with a Stallion Digital Workstation. The colocalized images with surface molecules pseudo-colored red and NSP4 pixels pseudo-colored green are shown in panels 3. Yellow pixels indicate the areas of pixel overlap. On the far right is shown the antibody controls.