Rabbit anti flag antibody

Forty-eight hours after transfection, cells were seeded on glass coverslips and then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked for 1 h in 2% BSA. Immunostaining was conducted with mouse anti-actin antibody (1 : 1000, ProteinTech) and rabbit anti-Flag antibody (1 : 200, ProteinTech) overnight at 4°C. Goat anti-mouse IgG (H+L) Alexa Fluor Plus 555 and goat anti-rabbit IgG (H+L) Alexa Fluor Plus 488 secondary antibodies (Invitrogen, USA) were used at 1 : 1000 at room temperature for 1 h. The cell nuclei were stained with DAPI (6-diamidino-2-phenylindole). Protein localization was observed by fluorescence microscopy (Carl Zeiss, Germany).

Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China). The Cell Counting Kit (CCK-8) was obtained from Biosharp (Anhui, China). Pyridone 6 (JAK inhibitor I) (A13457) was purchased from Adooq Biosciences (Irvine, CA, USA). Antibodies used for Western blotting, anti-phospho-STAT1 (S727), anti-STAT1, and anti-IFI35 antibodies (all rabbit polyclonal antibodies), were obtained from Abmart (Shanghai, China). Anti-IFIT2, anti-TLR3, anti-IRF7, anti-IRF3, anti-USP18, and anti-ISG15 antibodies (all rabbit) were purchased from ABclonal (Wuhan, Hubei, China). Anti-JAK1, anti-JAK2, anti-phospho-JAK1, and anti-phospho-JAK2 antibodies (all rabbit) were purchased from Abcam (Cambridge, MA, USA). Anti-MDA5 (IFIH1) and anti phospho-IRF3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-RIG-I (DDX58), anti-IFIT1, anti-IFIT3, anti-IFIT5, anti-IRF1, anti-IFITM1, and anti-β-actin antibodies were purchased from Proteintech (Chicago, IL, USA), and the antibodies used are listed in Additional file 7: Table S7. The monoclonal antibody against the JEV envelope (E) was kindly provided by Shengbo Cao (Huazhong Agricultural University, Wuhan, China). For immunofluorescence, rabbit anti-FLAG antibody was purchased from Proteintech.

Protein samples were separated on SDS-PAGE gels and then electroblotted onto nitrocellulose membranes. The nitrocellulose membranes were blocked for 1 h in 5% non-fat milk in TBST (10 mM Tris, pH 7.5, 200 mM NaCl, and 0.2% Tween 20) followed by incubation with primary antibodies: mouse anti-GFP antibody (Cat. 66002-1-Ig, Proteintech, Rosemont, IL, United States), rabbit anti-GFP antibody (Cat. ab290, abcam, Cambridge, United Kingdom), mouse anti-FLAG antibody (Cat. F3165, Sigma), rabbit anti-FLAG antibody (Cat. 20543-1-AP, Proteintech), rabbit anti-Myc antibody (Cat. 16286-1-AP, Proteintech), mouse anti-β-Actin antibody (Cat. 66009-1-Ig, Proteintech), rabbit anti-CDK2 antibody (Cat. 10122-1-AP, Proteintech), or rabbit anti-pCDK2 antibody (Cat. ab194868, abcam). Two secondary antibodies were used: goat anti-mouse antibody (Cat. ab216776, IRDye 680RD, abcam) and goat anti-rabbit antibody (Cat. ab216773, IRDye 800RD, abcam). Signals were captured by an Amersham Typhoon (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed by ImageQuant TL (GE Healthcare).