HEK293 cells were seeded in low density (50,000 cells per well) on a μ-Slide four-well chambered coverslip (ibidi, #80426) in DMEM supplemented with 10% FBS. The cells were cotransfected with differentially tagged variants of HRAS and NS1-VenusYFP using the TransFectin reagent (Bio-Rad, #1703352). Twenty-four hours after transfection, the cells were fixed for 10 min using 4% paraformaldehyde in PBS. The localizations of NS1 and NS1neg were recorded using an Axiovert 200M microscope (Carl Zeiss) with a 63-fold magnification. Acquisition was controlled with the VisiView4.0 software. The images were recorded over an exposure time of 200 ms and processed with the ImageJ software.

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