FIG. 4.

Effects of siRNA of hVAP-33 on the association of HCV NS proteins with detergent-resistant membrane fractions. (A) Immunoblotting analysis of protein expression level after transfection of siRNA against hVAP-33 or control siRNA in Huh7-replicon cells at 3 days posttransfection. Thirty micrograms of protein was loaded into each lane. Tubulin was detected as a loading control. Lanes: (−), untransfected; H2O, mock transfected. (B) Membrane flotation analysis. Huh7-replicon cells transfected with siRNA of hVAP-33 or control siRNA were harvested at 3 days posttransfection. Cell lysates were treated with 1% NP-40 for 1 h on ice and then fractionated by discontinuous sucrose gradient centrifugation, as detailed in Materials and Methods. NS proteins were analyzed by immunoblotting with individual monoclonal antibodies.