FIG 1.

(A) Identification of sorcin in microarray. Ab, antibody. (B) Sorcin interacts with NS5A protein. HEK293T cells were transiently transfected with a Myc-tagged NS5A plasmid. Total cell lysates were harvested and incubated with either purified GST or GST-tagged sorcin. GST-bound protein was detected by immunoblot analysis using an anti-Myc monoclonal antibody. (C) HEK293T cells were cotransfected with Myc-tagged NS5A and V5-tagged sorcin expression plasmids. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Myc monoclonal antibody, and then bound proteins were detected by immunoblot assay using an anti-V5 monoclonal antibody. Protein expression of Myc-tagged NS5A and V5-tagged sorcin was verified by immunoblotting with the indicated antibody. (D) NS5A protein interacts with the endogenous sorcin in HCV replicating cells. Huh7.5 cells were electroporated with 10 μg of Jc1 RNA. Cells lysates harvested at 4 days after electroporation were immunoprecipitated with either control serum or an anti-NS5A antibody. Bound protein was immunoblotted with an anti-sorcin antibody. Immunoprecipitation efficiency was verified by immunoblot analysis using an anti-NS5A antibody (lower panel). (E) Huh7.5 cells were either mock infected or infected with Jc1 for 4 h. At 48 h postinfection, cells were transfected with V5-tagged sorcin. At 24 h after transfection, cells were fixed in 4% paraformaldehyde, and immunofluorescence staining was performed by using an anti-V5 monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG to detect V5-tagged sorcin (green); a rabbit anti-NS5A antibody and TRITC-conjugated donkey anti-rabbit IgG were used to detect NS5A (red). Dual staining showed colocalization of sorcin and NS5A as yellow fluorescence in the merged image. Cells were counterstained with DAPI to label nuclei (blue). (F) Huh7.5 cells were either mock infected or infected with Jc1 for 4 h. At 48 h postinfection, cells were fixed in 4% paraformaldehyde, and immunofluorescence staining was performed by using an anti-sorcin monoclonal antibody and TRITC-conjugated donkey anti-mouse IgG to detect endogenous sorcin (red); a rabbit anti-NS5A antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG were used to detect NS5A (green). Dual staining showed colocalization of endogenous sorcin and NS5A as yellow fluorescence in the “Crop” image. IP, immunoprecipitation; IB, immunoblot; M1(R/G), Mander's colocalization coefficient (ratio of red and green fluorophore colocalization).