Figure 3

Surface distribution of wild-type and mutant F and G proteins. (A) Amino acid sequences of the cytoplasmic domains of wild-type and mutant F and G proteins. Numbers above the sequences indicate amino acid positions. Boldface letters indicate exchanged amino acid residues. Vertical lines indicate the beginning of the predicted transmembrane domains. (B and C) Surface distribution of wild-type F and G proteins in polarized endothelial cells. PAEC stably expressing either wild-type or mutant NiV F (B) or G (C) were grown on filter supports for 5 days and then incubated with a NiV-specific antiserum from the apical and basolateral sides without prior fixation. Surface-bound antibodies were detected with AlexaFluor 568-conjugated secondary antibodies. Confocal vertical sections through the cell monolayers are shown. (D) Cell surface proteins were labelled with S-NHS biotin from either the apical (ap) or the basolateral (bas) side. After cell lysis, F and G proteins were immunoprecipitated with NiV-specific antibodies. Precipitates were analyzed by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin and chemiluminescence.