Fig. S1. Cell surface distribution of CD81 and control proteins. Immunofluorescence images showing the basal membrane of living Huh-7 clones expressing EWI-2wint 1, LAL or Qcc. Experiments were performed at 37°C using TIRF microscopy. Cells were stained with Atto647N-labelled Fab fragments of TS81, SYB-1 and 11C5 mAbs to observe CD81, CD9 and CD46 respectively.


Fig. S2. EWI-2wint has no effect on CD46 dynamics. The distribution of all the apparent diffusion coefficients (ADC) was calculated for CD46 in Huh-7 clones expressing EWI-2, EWI-2wint (two clones), LAL or Qcc. Each dot represents one trajectory and 500 trajectories are represented for each cell clone. CD46 molecules were stained usingAtto647N-labelled Fab fragments of 11C5 mAb.


Fig. S3. EWI-2/EWI-2wint expression in CHO cells. CHO cells were stably transfected with hCD81 and either the empty vector (pcDNA3.1), the uncleavable EWI-2 (EWI-2, see Experimental procedures) or wild-type EWI-2 construct (EWI-2wint). It has to be noted that the cleavage of EWI-2 generating EWI-2wint naturally occurs in CHO cells. Cell surface biotinylation followed by immunoprecipitation assays were performed to control the expression of proteins. FLAG-tagged EWI-2 constructs and CD81 were immunoprecipitated with M2 and 5A6 mAbs respectively. Proteins were revealed by Western blotting with HRP-conjugated streptavidin. Asterisks indicate additional cleavage products of EWI-2wint, as previously described (Rocha-Perugini et al., 2008). Triangles indicate an unidentified partner of CD81, which is not related to EWI-2/EWI-2wint proteins.

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