Western Blot Analysis of Protein Targets

RIPA buffer (Sigma-Aldrich) plus a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) was used to lyse cells as previously described [18 (link)]. Proteins were loaded onto SDS-PAGE gels, transferred onto polyvinylidene difluoride membranes, and blocked with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at 4 °C. The following antibodies were used; Anti-FLAG tag (Wako), Anti-MYC tag (Abcam), Anti-GFP (Santa Cruz), Anti-Tubulin or β-actin antibodies (Abgent). Anti-pSTAT1/STAT1, Anti-HA tag, Anti-HIS tag, Anti-MYC tag, Anti-MDA5, Anti-RIG-I, Anti-pTBK1/TBK1, Anti-pIRF3/IRF3, Anti-MxA, Anti-OAS1, and Anti-RNaseL antibodies were from Cell Signaling Technology. Anti-SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (Sino Biological), while Anti-IFV NP antibody and Anti-ZIKV E antibody were purchased from GeneTex. The blots were washed with TBST for three times and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Proteins were visualized with ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Densitometry to quantify immunoblot bands was performed using ImageJ software.