Figure 3

Characterization of protein re-localization to sec-muNS-Mi* MS inside the ER promoted by IC*-tagging. (a) Immunofluorescence analysis of DF-1 cells transfected with sec-muNS-Mi* (sec-Mi* in the figure) and co-transfected either with sec-mRFP (upper row) or sec-mRFP-IC* (middle and lower row). mRFP fluorescence is seen in red, while sec-muNS-Mi* is shown in green after detection with muNS-specific antibodies. Nuclei were stained blue with DAPI and shown in the merged images. The white bar represents a distance of 5 µm. The pictures of the upper and middle row were taken with an epi-fluorescence microscope (epi on the left of the figure). The lower row shows pictures of a slice acquired with a confocal microscope. (b) Confocal microscopy analysis of DF-1 cells transfected with sec-muNS-Mi* and co-transfected with a chimera formed by the ectodomain of the glycoprotein of VSV fused to mRFP and the IC-Tag (VSV-G). The presence of sec-muNS-Mi* was revealed by using muNS-specific antibodies (green). The VSV chimera was detected by the mRFP fluorescence. Nuclei were stained blue with DAPI and shown in the merged image. The white bar represents a distance of 5 µm. (c) Western-blot analysis performed with a mix of antibodies against the SV5 epitope and muNS. The samples came from extracts from DF-1 cells transfected with expression plasmids for a VSV chimera similar to the one described in b, but where the mRFP was replaced by the SV5 epitope (1 and 2) or co-transfected with sec-muNS-Mi* (3 and 4) either before (1 and 3) or after (2 and 4) treatment with N-glycosidase.