Protein concentrations of whole brain lysates were estimated by Bradford method. Sample volumes containing 20 mg of protein were electrophoresed on polyacrylamide gel and transferred onto nitrocellulose membrane. After blocking with 7% skimmed milk, the blots were incubated overnight at 4uC with primary antibodies against JEV E-glycoprotein (Abcam, USA), and JEV NS5 (a kind gift from Dr. Chun-Jung Chen, Taichung Veterans General Hospital, Taichung, Taiwan), iNOS (Upstate-Chemicon, USA), HSP-70, SOD-1 (Santa Cruz Biotechnology, CA, USA), TRX (AB Frontiers, Korea; a kind gift from Dr. Ellora Sen, NBRC), phospho NFkB, phospho ERK1/2, total ERK1/2 and phos-phoP38 MAP kinase (Cell Signaling, USA) at 1:1000 dilutions. After extensive washes with PBS-Tween, blots were incubated with appropriate secondary antibodies conjugated with peroxidase (Vector Laboratories, CA, USA). The blots were again washed with PBS-Tween and processed for development using chemiluminescence reagent (Millipore, USA). The images were captured and analyzed using Chemigenius, Bioimaging System (Syngene, Cambridge, UK). The blots were stripped and reprobed with antib-tubulin (Santa Cruz Biotechnology, USA) to determine equivalent loading of samples [22] .