Anti s tag

Cell lysates were harvested in NP-40 lysis buffer containing protease inhibitor cocktail (Roche Applied Bioscience) and quantitated using a Pierce bicinchoninic acid protein assay kit (Fisher Scientific). Equivalent amounts of protein were separated in Mini-Protean TGX precast 4 to 20% gels (Bio-Rad Laboratories, Hercules, CA, United States) and transferred to nitrocellulose membranes. Membranes were blocked in phosphate-buffered saline (PBS) containing 5% milk and 0.1% Tween 20 and incubated with primary antibody. The following antibodies were used: anti-S-tag (1:1000; Abcam, Cambridge, MA), anti-UBR5 (1:1000; Cell Signaling Technology, Danvers, MA, United States), anti-HBZ (1:1,000), anti-APH-2 (1:1,000), anti-FLAG clone M2 (1:1,000; Agilent Technologies, Santa Clara, CA, United States), anti-ubiquitin (1:250; Santa Cruz Biotechnology), anti-β-actin (1:5,000; Sigma–Aldrich), and anti-α-Tubulin (1:250; Santa Cruz Biotechnology). The secondary antibodies used were horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse immunoglobulin antibodies (1:5,000; Santa Cruz Biotechnology). The blots were developed using an ECL Western Blotting Substrate (Fisher Scientific). Images were taken using an Amersham Imager 600 imaging system (GE Healthcare Life Sciences), and densitometric data were calculated using the ImageQuant TL program (GE Healthcare Life Sciences).

Rabbit antisera to SFTSV N, GP (GN), or RdRp were described previously [15 (link), 17 (link)]. Mouse anti-N serum was raised against SFTSV N protein prepared from Escherichia coli. Mouse anti-Flag (Sigma-Aldrich, Cat#F3165), anti-HA (Sigma-Aldrich, Cat#05–904) and anti-IFNAR1 (Sigma-Aldrich, Cat#SAB1406003) antibodies and rabbit anti-MOV10 (Proteintech, Cat#10370-1-AP), anti-β-actin (ABclonal, Cat#AC026), anti-Stag (Abcam, Cat#ab18588) and anti-GFP (Abcam, Cat#ab6556) antibodies were purchased from the indicated manufacturers. For the secondary antibodies, goat anti-mouse IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, Cat#A-11001) and goat anti-rabbit IgG conjugated with Alexa Fluor 647 (Thermo Fisher Scientific, Cat#A32733) were used in the IFA assay; goat anti-mouse or anti-rabbit IgG antibodies conjugated with HRP (Abcam, Cat#ab6789 and Cat#ab6721) were used for IB analysis.