Nb300 605

Protein lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer with protease (P8340; Sigma-Aldrich) and phosphatase inhibitors (4906845001; Roche, Basel, Switzerland). Sixty-five micrograms was electrophoresed on a 4–15% Mini-Protean^® TGX Stain-Free^™ precast gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a 0.45 μm PVDF membrane (Bio-Rad Laboratories) by wet transfer. The membrane was then blocked [5% bovine serum albumin (BSA) diluted in Tris-buffered saline with 0.1% Tween (TBST)] for 1 hour, followed by overnight incubation at 4 °C with the primary antibody. Primary antibodies used were: (i) anti-iNOS (1:1000, NB300-605; Novus Biologicals, Littleton, CO USA); and (ii) anti-eNOS (1:1000, PA1-037; Invitrogen). The membrane was then incubated with the corresponding secondary antibody (infrared fluorescent-conjugate) for 1 hour at room temperature. Blots were scanned on the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE USA). Densitometry was quantified using Image Studio Lite (v5.2.5, LI-COR Biosciences) and ImageLab^™ Software (v6.0.1, Bio-Rad Laboratories), and normalized against total protein through stain-free imaging technology (Bio-Rad Laboratories) [36 (link), 37 (link)].

RAW 264.7 macrophages were seeded at the density of 5 × 10⁵ cells per well over coverslips in 6-well plates, activated as described above. Cells were fixed for 30 min with PBS containing 4% formaldehyde and washed three times with PBS. Non-specific binding was blocked by treatment of cells with bovine serum albumin (BSA, 4%; Thermo Fisher, Waltham, MA, USA), diluted in PBS for 1 h at room temperature. M1 cells were incubated with iNOS rabbit Polyclonal antibody (NB300-605, 1:200, Novus Biologicals) at 4 °C overnight, and M2 cells were incubated with F4/80 mouse monoclonal antibody, Alexa Fluor 488 (1:200, Catlog #53-4801-82, Thermo Fisher) and CD209 Rabbit mAb (1:200, A9649, ABclonal, Woburn, MA, USA) at 4 °C overnight. Secondary antibodies included goat anti-mouse IgG1 cross-adsorbed secondary antibody, Alexa Fluor 488 (1:500, Catalog# A-21121, Thermo Fisher), and goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594 (1:500, Catalog# A11037, Thermo Fisher). Coverslips containing cells were mounted with the Prolong Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA, USA), visualized under a fluorescence microscope (Leica, DM 6000B, Werzlar, Germany) at 400× magnifications, and analyzed with Image J (NIH, Bethesda, MD, USA).

Mouse monoclonal antibodies against E, NS1, and NS1′ of JEV were generated in our laboratory. BALB/c mice were immunized with synthetic NS1 amino acid peptide or ΔNS1′ amino acid peptide (52 amino acids generated by −1PRF). Anti-NS1 or NS1′ monoclonal antibody was screened by the hybridoma cell fusion technique and indirect enzyme-linked immunosorbent assay (ELISA). Commercial antibodies used in this study are listed as follow: JEV NS3 protein rabbit polyclonal antibody (GTX125868; GeneTex), CD11b rabbit monoclonal antibody (ab133357; Abcam), CD163 rabbit monoclonal antibody (ab182422; Abcam), MHC II (also known as SLA) mouse monoclonal antibody (MCA2314GA; Bio-Rad), iNOS rabbit polyclonal antibody (NB300-605; Novus), CD172a mouse monoclonal antibody (NBP2-61014; Novus), TNF-α goat polyclonal antibody (AF690; R&D Systems), GAPDH mouse monoclonal antibody (AC033; ABclonal), goat anti-rat IgG Alexa Fluor 555 (A-21434; Invitrogen), goat anti-rabbit IgG Alexa Fluor 488 (A-11008; Invitrogen), goat anti-mouse IgG-horseradish peroxidase (HRP) (31430; Invitrogen), and goat anti-mouse IgG-HRP (31430; Invitrogen).

Primary antibodies used for immunoblotting: NOS2 (NB300-605, 1:1000, Novus, Littletown, CO, USA); NOS3 (ab5589, 1:1000, Abcam, Cambridge, MA, USA); FLG (NBP1-87527, 1:2000, Novus, Littletown, CO, USA and PRB-417P-100 1:1000, Covance, Princeton, NJ, USA); LAMIN A/C (4777, 1:5000, Cell Signaling Technology, Danvers, MA, USA), ACTB (A5441, 1:100,000, MilliporeSigma, Billerica, MA, USA). The HRP-conjugated secondary antibodies were used for immunoblotting: goat anti-rabbit (111035144, 1:10,000, Jackson ImmunoLaboratories Research, West Grove, PA, USA) and goat anti-mouse (115035003, 1:10,000, Jackson ImmunoLaboratories Research, West Grove, PA, USA).

Briefly, cultured macrophages were fixed in 4% formaldehyde for 1 h and incubated with a rabbit anti-iNOs antibody (M1 polarization marker, 1:200, NB300-605, NOVUS Biologicals, Building IV Centennial, CO 80112, USA) or rabbit anti-Arg-1 antibody (M2 polarization marker, 1:200, NBP1-32731, NOVUS Biologicals, Building IV Centennial, CO 80112, USA) overnight at 4°C. The cells were then PBS-washed and incubated with fluorescein isothiocyanate FITC/TRITC-conjugated goat anti-rabbit secondary antibodies (1:50, ZF0311; ZF0316, Zhongshan JinQiao Biotechnology Co., Ltd, Beijing, China). After washed with PBS, the cells were stained with DAPI (40 mg/ml) for 5 min and examined with a laser-scanning microscope (ZEISS, Germany). The IODs of the target proteins were analyzed using the Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD). Briefly, a Leica DMIRB microscope was used to examine each cell section, and 200× non-overlapping high-power fields (0.5 × 0.5 mm, total area of 0.25 mm²) in the center of the eyepiece were selected. The IODs in the regions of interest were measured as gray values, and the number of positive cells in these regions was counted. Three fields within the regions of interest in three slices from each group were examined. For each slice, the ratios of the IOD to the number of positive cells in three selected fields were averaged.