Anti grp78

WNV_KUN specific anti-NS1 (clone 4G4; (12)), anti-NS5 (clone 5H1.1) and anti-Envelope monoclonal antibodies were generously provided by Dr Roy Hall (University of Queensland, Brisbane, Australia). WNV_KUN-specific rabbit anti-NS3 polyclonal antisera has been described previously (18). Rabbit anti-Calnexin was purchased from Epitomics. Rabbit anti-Actin and anti-GRP78 were purchased from Sigma. Rabbit anti-PLA2G4A and anti-phosphorylated PLA2G4A were purchased from Cell signaling Technology, and anti-PLA2G4C from Novus Biologicals. Mouse anti-dsRNA (clone J2) antibodies were purchased from English & Scientific Consulting Bt. (Hungry). Alexa Fluor 488- and 594-conjugated anti-rabbit and anti-mouse specific IgG were purchased from Molecular Probes (Invitrogen, Leiden, The Netherlands).

Protein was extracted from the cultured cells using RIPA buffer. A total of 30 μg proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBST containing 5% skimmed milk, and incubated with the primary antibodies including: anti-GRP78 (1:1000; Sigma Aldrich), anti-PERK (1:1000; Sigma Aldrich), anti-p-PERK (1:500; Sigma Aldrich), anti-eIF2α (1:1000; Abcam, Cambridge, UK), anti-p-eIF2α (1:500; Abcam), anti-ATF4 (1:1000; Sigma Aldrich), anti-CHOP (1:500; Sigma Aldrich), anti-SIRT1 (1:1000; CST, Beverly, USA), anti-Bcl-2 (1:1000; Proteintech, Wuhan, China), Bax (1:1000; Proteintech), anti-Mcl-1(1:1000; Sigma), anti-caspase 3 (1:1000; CST), anti-caspase 9 (1:1000; CST), anti-cleaved caspase 3 (1:500; Sigma Aldrich), and anti-β-actin (1:1000; Origene, Rockville, USA), followed by incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, chemiluminescent substrate (Beyotime) was used for signal visualization and detection. The density of each band was normalized to its respective loading control (β-actin). The immunoblots were quantified by densitometric analysis.

bEnd.3 cells were fixed in 4% paraformaldehyde for 15 min and then incubated in 5% bovine serum albumin with 0.1% Triton X-100 for 1 h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the anti-GRP78 (1:1,000; Sigma, United States) overnight at 4°C. Alexa Fluor 594-conjugated goat anti-rabbit IgG polyclonal antibody (1:200, Cell Signaling, MA, United States) was used as the secondary antibody. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Immunofluorescence results were visualized by using a Zeiss Imager Z2 microscope (Carl Zeiss, Oberkochen, Germany). The average fluorescence intensity was analyzed using Image J (v1.8.0.112).