FIG 8.

Calcium-binding activity of sorcin is required for HCV propagation. (A) HEK293T cells were cotransfected with a Myc-tagged NS5A and V5-tagged wild-type or mutant sorcin plasmid. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Myc monoclonal antibody, and bound proteins were immunoblotted with an anti-V5 antibody. Immunoprecipitation efficiency was verified by immunoblotting with an anti-Myc monoclonal antibody using the same lysates. (B) Huh7.5 cells were transfected with the indicated siRNAs. At 24 h after siRNA transfection, cells were transfected with the indicated combinations of expression plasmids and then infected with Jc1 for 4 h. At 48 h postinfection, cell lysates were immunoblotted using the indicated antibodies. Band intensities of HCV NS5A protein were normalized against those of β-actin. Pos, HCV-specific siRNA targeting the 5′ nontranslated region (NTR) of Jc1; Neg, universal negative-control siRNA; V5-sorcin-Res, V5-tagged siRNA-resistant sorcin mutant; V5-sorcin-Res+F112L, V5-tagged siRNA-resistant calcium-binding-defective sorcin mutant.