C7 50

Western blotting was performed mainly as described previously [36 (link)]. Briefly, cell lysates in reducing sample buffer were electrophoresed in SDS-12% polyacrylamide gels under reducing conditions, in parallel with protein molecular mass standards (MagicMark^™, Invitrogen). Subsequently, proteins were transferred onto nitrocellulose membranes (Amersham Protran, GE Healthcare). Membranes were blocked with PBS containing 0.05% Tween 20 and 2% ECL Prime Blocking Agent (GE Healthcare), and probed 1h with primary antibodies directed against HCV core protein (C7-50, Alexis Biochemicals), HCV E2 glycoprotein (AP33, kindly provided by A. H. Patel, MRC Virology Unit, Institute of Virology, Glasgow, UK), HCV NS3 protein (8 G-2, Abcam), or calnexin (C4731, Sigma-Aldrich). Excess antibody was removed with five washes, and the second-step antibody (DyLight 800 labeled goat anti-mouse or DyLight 680 labeled goat anti-rabbit, KPL) diluted 1:15,000 in blocking buffer was allowed to bind for 1 h. After several washes, protein bands were visualized using LI-COR-Odyssey infra-red scanner (LI-COR).

Western blotting for SR-BI, CLDN1, OCLN, HCV nonstructural protein 3 (NS3) and HCV core protein was performed as previously described [12 (link),19 (link)]. Anti-SR-BI polyclonal antibody (Abcam) and monoclonal antibodies against CLDN-1 (Zymed, San Francisco, CA, USA), OCLN (ABCYS, Paris, France), HCV core protein (C7-50; Alexis Biochemicals, San Diego, CA, USA), NS3 (1847; Virostat, Portland, MA), or alpha-actin (Cedarlane Laboratories Ltd, Ontario, Canada) were used as primary antibodies.