Figure 3: ARFRP1 specifically interacts with NS5A.

(A) HEK293T cells were transfected with the indicated combinations of plasmids. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Myc monoclonal antibody, and then bound proteins were immunoblotted by using an anti-Flag antibody (lower, top panel). Immunoprecipitation efficiency of each viral protein was verified by immunoblotting with an anti-Myc antibody (lower, bottom panel). IP, immunoprecipitation; IB, immunoblot. Input proteins were verified using indicated antibodies (upper panels). Asterisks indicate the heavy and light chain IgG. All experiments were carried out in duplicate. (B) HEK293T cells were cotransfected with Flag-tagged AFRRP1 and Myc-tagged NS5A (genotype 1b or 2a). At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Myc antibody, and then bound proteins were detected by immunoblot analysis using an anti-Flag antibody (upper panel). Protein expressions of Myc-tagged NS5A (lower, left panel) and immunoprecipitation efficiency (lower, right panel) were verified using an anti-Myc antibody. (C) Schematic illustration of both wild-type and mutant constructs of NS5A plasmid. (D) HEK293T cells were cotransfected with the indicated combinations of expression plasmids. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Myc antibody, and bound proteins were immunoblotted with an anti-Flag antibody (upper, right). Both protein expressions and immunoprecipitation efficiency were verified by immunoblotting with the indicated antibodies (lower panels). (E) (Upper, top panel) Huh7.5 cells infected with Jc1 were immunoprecipitated with either IgG or an anti-ARFRP1 antibody, and bound protein was immunoblotted with rabbit anti-NS5A antibody. Arrowhead indicates NS5A and arrow denotes ARFRP1. (Lower, top panel) The same cell lysates were immunoprecipitated with either control rabbit serum or rabbit anti-NS5A antibody, and bound protein was immunoblotted with an anti-ARFRP1 antibody. Arrowhead indicates ARFRP1 and arrow denotes NS5A protein. (F) Huh7.5 cells were transfected with Flag-tagged ARFRP1 plasmid and then infected with Jc1. Immunofluorescence staining was performed by using rabbit anti-NS5A antibody and TRITC-conjugated goat anti-rabbit IgG to detect NS5A (red), and an anti-Flag antibody and FITC-conjugated goat anti-mouse IgG to detect ectopic expression ARFRP1 (green). Cells were counterstained with DAPI to label nuclei (blue). (G) Huh7.5 cells were either mock-infected or infected with Jc1. Immunofluorescence staining was performed by using rabbit anti-NS5A antibody and TRITC-conjugated goat anti-rabbit IgG (red), and an monoclonal anti-ARFRP1 antibody and FITC-conjugated goat anti-mouse IgG to detect endogenous ARFRP1 (green).