Cl casp3

The following antibodies against Flavivirus E protein (1:200, mouse, Millipore, MAB10216), J2 double-stranded RNA (1:300, mouse, English and Scientific Consulting Kft, Hungary), Myelin Protein Zero (P0) (1:500, chicken, AVES labs, PZO), Neurofilament-H (1:200, rabbit, Millipore, AB1989), CHOP (1:600, mouse, 2895 S, Cell Signaling) and cl-CASP3 (1:300, rabbit, 9661, Cell Signaling) were used. Goat Anti-mouse FITC (1:200, SouthernBiotech, 1034–02), donkey anti-chicken TRITC (1:200, Jackson Immunoresearch, 703–025–155) and donkey anti-rabbit Cy5 (1:200, Jackson Immunoresearch, 711–175–152) were used as secondary antibodies.

Equal amounts of protein were separated via electrophoresis on NuPAGE 4-12% gradient precast polyacrylamide gels (Life Technologies, Carlsbad, CA). Proteins were transferred onto Hybond-P PVDF membranes (Millipore, Billerica, MA) and blocked for 1 h at room temperature in 5% non-fat milk prepared in Tris Buffered Saline with 0.1%Tween-20 (vol/vol). Membranes were incubated with primary antibodies overnight at 4 °C. Primary antibody concentrations were as follows: GRP78 (1:1000; Cat#3177), Cl CASP3 (1:250; Cat#9664), GADD153 (1:500; Cat#5554), Cl PARP (1:1000; Cat#9541), ATF4 (1:500; Cat#11815), p-eIF2α (1:500; Cat#3398), NF-κB (1:1000; Cat#8242), XIAP (1:250; Cat#2042), and PDI (1:5000; Cat#3501) were obtained from Cell Signaling Technologies; XBP1s (1:500; Cat#37152) was obtained from Abcam; ATF6 (1:500; Cat#24169-1-AP) was obtained from Proteintech; MCL1 (1:1000; Cat#sc-819) was obtained from Santa Cruz Biotechnology; and NCOA3 (1:5000; Cat#PA1-845) was obtained from ThermoScientific. Following primary incubation, immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced-chemiluminescence detection system (ThermoScientific, Rockford, IL). Immunoreactive band density was then quantified using ImageJ software.