Immunoprecipitation.

Immunoprecipitations were performed with JEV-infected (MOI, 10) Neuro2a/Huh-7 cells plated in 100-mm dishes. At 24 h p.i., the cells were lysed by adding 0.5 ml lysis buffer (20 mM Tris HCl, 1 mM EDTA, 250 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM protease inhibitor). The total cell lysate was precleared with protein G or protein A beads. This was followed by addition of 5 μg JEV E (ab81193)-mouse IgG or 10 μg GRP78 (ab32618)-rabbit IgG antibodies for 6 to 8 h, followed by immobilization with protein G or protein A beads, respectively. Bound proteins from the beads were eluted using SDS sample buffer and analyzed by gel electrophoresis and Western blotting.