Mouse anti flag

Human embryonic kidney 293T (HEK293T) cells, HeLa cells, COS-7 cells, and Vero cells were grown in Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL of penicillin and streptomycin at 37°C in a 5% CO₂ incubator. Mouse anti-Flag, anti-Myc, anti-GFP, and anti-hemagglutinin (HA) monoclonal antibodies (MAbs) were acquired from Abmart (Berkeley Heights, NJ, USA). IgG negative-control antibody was purchased from Proteintech (Wuhan, China). Rabbit anti-β-actin MAb was bought from ABclonal (Wuhan, China). Rabbit anti-IKKi, anti-TBK1, anti-IRF3, anti-phosphorylated (phospho)-IRF3 (Ser396), and anti-phospho-TBK1 (Ser172) MAbs, alkaline phosphatase (AP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were provided by Cell Signaling Technology (Boston, MA, USA). Cyanine 5 (Cy5)-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG were supplied by Bioss (Beijing China) and BBI Life Sciences, respectively.

FreeStyle 293 F cells cultured in FreeStyle 293 medium were transfected with indicated plasmids (Fig. 5f) at a ratio of 1:1 using PEI. Forty-eight hours post-transfection, cells were collected and sonicated in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 1 mM PMSF. After centrifugation at 16,162 × g for 20 min, the supernatants were mixed with Glutathione Sepharose 4B (GE Healthcare) and incubated at 4 °C for 1 h. Then the beads were washed with 20 mM Tris (pH 7.5) and 150 mM NaCl six times. Bound proteins were eluted with 50 mM Tris (pH 8.0), 150 mM NaCl, and 10 mM reduced glutathione.
For Western blot, proteins were separated with 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked using 5% nonfat milk in TBST for 1 h at room temperature and incubated with primary antibodies [mouse anti-GST (30901ES10; Yeasen Biotechnology) or mouse anti-FLAG (M20008M; Abmart), both antibodies were diluted 5000 times] overnight at 4 °C. Then membranes were incubated with horseradish-peroxidase (HRP) labeled goat anti-mouse secondary antibody (31444; Thermo Fisher Scientific, the antibody was diluted 10,000 times) for 1 h at room temperature and developed using High-Sig ECL Western Blotting Substrate (Tanon).

Mouse anti-Flag, anti-Myc, anti-hemagglutinin (HA), and anti-β-actin monoclonal antibodies (mAbs) were purchased from ABmart. Rabbit anti-Flag polyclonal antibody (pAb) was purchased from Proteintech. Mouse non-specific control IgG antibody was purchased from eBioscience Inc. Rabbit anti-gD pAb was gifted by Dr. Roselyn J. Eisenberg (School of Veterinary Medicine, University of Pennsylvania), and mouse anti-gD mAb was purchased from Santa Cruz Biotechnology.