Figure 3
Positive FRET analyses by acceptor photobleaching between NSP4 and surface proteins of RV-infected MDCK and HT29.F8 cells. Live cells were surface biotinylated prior to fixation and permeabilization. Streptavidin-CY3 was utilized to label all surface biotinylated proteins of the exofacial PM including NSP4, if exposed. NSP4 was labeled with Cy5-linked F(ab)2 prepared from NSP4150-175 rabbit antisera. A. CY5 (NSP4) and CY3 (PM) images were acquired with the 647-nm and 568-nm-wavelength lasers both prior (merged CY3 and CY5 images, panels 1,4,7) and after (same cells, panels 2,5,8) photobleaching of the NSP4 CY5 (donor) signal in infected MDCK cells at 4 hpi (panels 1,2,3) and 7 hpi (panels 4,5,6). RV-infected HT29.F8 cells (MOI = 2) were examined at 7 hpi (panels 7,8,9). FRET was calculated from the fluorescence intensity of selected areas of the PM (yellow box) both before and after bleaching. Panels 3, 6, 9 show a magnification of the photobleached regions of the PM delineated by the yellow boxes in panels 1, 4, and 7. Panels were pseudo-colored to show relative intensities with its respective calculated FRET values. Both the pre (top panels of 3, 6, 9) and post bleaching (bottom panels 3, 6, 9) images are shown. Values were calculated from 10 different regions taken from two separate experiments for each time point. Controls for each region were calculated from a region of PM in which NSP4 was not present and was calculated at 0% FRET. Vertical bar = relative intensity scale with red > green > blue. B shows an example of a cell that lacked membrane integrity (*) and therefore intracellular molecules were biotinylated and bound CY3. Cells showing intracellular staining with CY3 were not used.