Nfkb1

1x10⁶ PBMCs/ml were incubated in 24 well- plates with 50 ng/ml of recombinant human IFN-γ (PeproTech) for 0 (at rest), 30, 120 and 180 min in complete RPMI medium. To ensure equal loading, protein concentrations were assessed via Pierce™ BCA Protein Assay Kit (Thermo Scientific). Lysates were denatured in 1x LDS buffer (Invitrogen) then boiled for 10 mins at 70°C. Lysates were then run on NuPAGE Novex 4-12% gels (Invitrogen) with Spectra Multicolour Broad Range Protein Ladder (Invitrogen). Electrophoresis was run a 350A for 1 hour to transfer protein to a PDVF membrane. Membranes were then blocked with 5% non-fat milk in 1x Tris buffered saline with 0.1% Tween20 (Sigma) (TBST) for 1 hour at room temperature. Primary antibodies used were B-actin, Phospho-STAT1, STAT1, pJAK1, STAT3, pSTAT3, Cyclophilin B, NFKB1, pAKT, AKT SOCS1, FAK, and JAK2 (all Cell Signaling). Membranes were incubated overnight with primary antibodies, then washed 3x 10mins with TBST, before being incubated with a rabbit HRP-conjugated secondary antibody for 1 hours at room temperature. Membranes were washed a further 3x 10mins with TBST then developed with chemiluminescence ECL or Femto (Thermo Fisher).

Cells were collected and boiled for 10 min in the RIPA buffer (Sigma). 20 μg protein were resolved on SDS-PAGE gels and electro-transferred onto nitrocellulose membranes (Millipore, Merck, Shanghai, China) using a power supply (BIO-RAD, Guangzhou, China) at 250 mA for two h. Membranes were blocked by non-fat milk. Antibodies: p65 (Cell Signalling Technology, Shanghai, China; 6956, 1:1000), p-p65 (Ser536, Cell Signalling Technology, 3033, 1:1000), EZH2 (Cell Signalling Technology, 5246 , 1:1000), H3K4me3 (Cell Signalling Technology, 9751, 1:1000), H3K9me3 (Cell Signalling Technology, 13969; 1:1000), H3K36me3 (Cell Signalling Technology, 4909, 1:1000), H3K27me3 (Cell Signalling Technology, 9733, 1:1000), H3K27me2 (Cell Signalling Technology, 9728, 1:1000), H3K27me1 (Cell Signalling Technology, 84932, 1:1000), H3 (Abcam, Hangzhou, China; Ab1791, 1:10,00), ACTB(Cell Signalling Technology, 4970, 1:1000), NFKB1(Cell Signalling Technology, 13586, 1:1000), NFKB2 (Cell Signalling Technology, 37359, 1:1000) and GAPDH (Cell Signalling Technology, 97166, 1:3000). The blots were covered with respective primary antibodies at cold room and corresponding secondary antibodies (1:3000, ThermoFisher, USA). The membranes were processed using an ECL kit (ThermoFisher, USA).

Protein was extracted from GC cell lines and paired primary tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA) and 20 μg of protein mixed with 2 × SDS loading buffer was loaded per lane, separated by 12 % SDS-polyacrylamide gel electrophoresis. The primary antibodies used in this study includes NFKB1 (#3035, Cell Signaling), RELA (#3034, Cell Signaling), p21 (#2946, Cell Signaling), p27 (#2552, Cell Signaling), p-Rb (Ser807/811) (#9308, Cell Signaling), cleaved-PARP (Asp214) (#9541, Cell Signaling) and GAPDH (#2118, Cell Signaling). The secondary antibodies were anti-Mouse IgG-HRP (00049039, Dako, 1:30000) and anti-Rabbit IgG-HRP (00028856, Dako, 1:10000). The Western blot bands were quantified by ImageJ.

Vero cells were infected with either PEDV strains CT-P10 or CT-P120 at an MOI of 0.1, and then lysed using cell lysis buffer (Beyotime, Shanghai, China). The lysates were subjected to boiling, following which the proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). The resolved proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking non-specific binding sites, the membrane was incubated overnight at 4 °C with primary antibodies against NFKB1 (Proteintech, San Diego, CA, USA, 14220-1-AP), ISG15 (Affinity, Melbourne, Australia, AB2838282), IRF3 (Proteintech, 11312-1-AP), TRAF3 (Cell Signaling Technology, Danvers, MA, USA, 4729S), PEDV-N, and GAPDH (Cell Signaling Technology) to detect respective proteins. Subsequently, the blots for NFKB1, ISG 15, IRF3, TRAF3, and GAPDH were incubated with HRP-labeled anti-rabbit IgG secondary antibody (Cell Signaling Technology), whereas the PEDV-N blot was incubated with HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology). The protein bands were visualized using the ECL Plus chemiluminescent substrate (Pierce, Rockford, IL, USA).