Figure 9
Western Blot of surface biotinylated, uninfected MDCK and HT29.f8 cells in which media containing soluble NSP4 was exogenously added. RV-free, NSP4-containing media exogenously was added to MDCK or HT29.F8 naïve cells and incubated for 90 min at 4°C. The treated cells were washed, surface biotinylated, lysed and the surface molecules precipitated with streptavidin agarose. The surface precipitates and lysates were separated by SDS-PAGE and analyzed by Western blot using anti-NSP4150-175. Dimeric and monomeric NSP4 are indicated. Lanes are as follows: 1 = MDCK lysates following the streptavidin pull-down. 2 = MDCK streptavidin pull-down (exofacial PM). 3 = MDCK cell lysates from the no biotin control. 4 = MDCK streptavidin pull-down from the no biotin control. 5 = Uninfected MDCK cell lysates. 6 = HT29.F8 lysates following the streptavidin pull-down. 7 = HT29.F8 streptavidin pull-down (exofacial PM). 8 = HT29.F8 cell lysates from the no biotin control. 9 = HT29.F8 streptavidin pull-down from the no biotin control