Odyssey infrared imager

Protein spots were visualized by staining with Coomassie Brilliant Blue G-250. Gel images were captured by LI-COR odyssey infra-red imager (LI-COR Biosciences, USA). Four biological replicates each with two analytical replicate (n = 8) images per dataset (mock-infected control versus JEV infected) were used for automatic spot detection using PD Quest 2D Analysis Software (Hercules, CA, USA). Spot intensities were normalized by total valid spot intensities and mean of values from duplicate analytical gels from four biological replicates were subjected to paired t-test analysis using GraphPad Prism software. Protein spots showing altered expression between control and experimental groups (|ratio|≥1.5, p≥0.05) were marked and excised.

Whole cell lysates were collected in lysis buffer containing 1x protease inhibitor cocktail. Protein concentration was measured by the BCA protein assay (Thermo Scientific, Rockford, IL), separated by electrophoresis through 4–12% or 7.5% SDS-PAGE and then transferred to Immobilon-FL PDVF membranes (Millipore, Billerica, MA). Membranes were blocked in LiCor blocking buffer (LiCor, Lincoln, NE) diluted with PBS (1:1), then incubated with primary antibodies overnight at 4°C. Following extensive washing, membranes were incubated with species-specific IRDye700 or 800-conjugated secondary antibodies (1:15,000) for 1 h at room temperature, and visualized with a LiCor Odyssey infrared imager (LiCor, Lincoln, NE). Consistent protein loading was determined by reprobing membranes with anti-actin or anti-GAPDH antibodies. To probe with lectins, the protein blot was blocked with protein-free blocking buffer (PFB, Thermo Scientific, Rockford, IL) at room temperature for 1 h, and then probed overnight at 4°C with 1 μg/ml of biotinylated lectins (Vector Laboratories, Burlingame, CA) in lectin-binding buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 0.1 mM CaCl₂). The filters were then washed with TBST (TBS with 0.05% Tween-20), incubated with IRDye 800-conjugated streptavidin in PFB for 1 h and then washed with TBST. The blots were imaged on the LiCor Odyssey system.

After the indicated treatments, whole cell lysates were prepared in ELB buffer (50 mM HEPES (pH 7), 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 10 mM NaF, 0.1 mM Na₃VO₄, 50 μM ZnCl₂, supplemented with 0.1 mM PMSF, 1 mM DTT, and a mixture of protease and phosphatase inhibitors). After removal of cellular debris via high-speed centrifugation, lysates were fractionalized on 7.5% SDS PAGE gels and protein was electrophoretically transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, USA). The membranes were then analyzed for immunoreactivity against the following antibodies: rabbit anti human total p38, mouse anti human phosphorylated p38, and phosphorylated activating transcription factor2 (ATF-2) (Cat# 9212s, Cat# 9216s, Cat# 9221; all from Cell Signaling Technology, Danvers, MA, USA). Bound antibodies were detected using species-specific infrared dye (IR-dye) conjugated secondary antibodies and subsequently developed using the LICOR Odyssey Infrared Imager (LICOR Biosciences, Lincoln, NE, USA).