Fig 7.

Epifluorescence analysis of proximity ligation assays. Cells harboring the subgenomic replicon I389-neo/NS3-3′/5.1 were analyzed by epifluorescence microscopy, as described in Materials and Methods, using an anti-NS5A antibody (antibody [Ab] 1877, IgG2A isotype, from AbD Serotec) in conjunction with anti-mouse Alexa Fluor 594-conjugated secondary antibody (red). ER (calnexin) or nuclear (ASH2L, IgG2A isotype) protein markers were detected using an anti-rabbit Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). (A) ASH2L expression was localized in all cell nuclei. A proportion of NS5A was shown to colocalize with ASH2L in the nucleus. (B) As shown in the merged image, NS5A mainly colocalized with ER, except for some spots present in the nucleus. Cells harboring the subgenomic replicon I389-neo/NS3-3′/5.1 were analyzed by epifluorescence microscopy after proximity ligation assays (Duolink InSitu). (C) Proximity ligation was performed using a mouse anti-NS5A antibody (OBT 1222) and a rabbit anti-NS5A antibody (29) as positive controls to confirm the expression of NS5A in this cell line. In SGR-harboring cells, the fluorescent dots were mainly localized in the cytoplasm, with a small proportion in the nucleus. (D) Proximity ligation was performed using a mouse anti-NS5A antibody (OBT 1222) and a rabbit anticalnexin antibody. This analysis confirmed the presence of NS5A in the cytoplasm, in close proximity to ER proteins (calnexin). (E) Proximity ligation was performed using a mouse anti-NS5A antibody (OBT 1222) and a rabbit anti-ASH2L antibody. This analysis showed the presence of a proportion of NS5A proteins in close proximity to nuclear proteins in the nucleus. (F) As a negative control, proximity ligation was performed using PLA probes only. Nuclei were visualized using To-Pro-3 staining.