Nucleocapsid protein

In general, we added NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) to a 1× concentration and incubated samples for 3 min at 95 °C. Proteins were resolved by SDS–polyacrylamide gel electrophoresis using NuPAGE 4 to 12% Bis-Tris-HCl Gels (Thermo Fisher Scientific) at 200 V for 1 h, followed by transfer to a nitrocellulose membrane using the iBlot dry blotting system (Thermo Fisher Scientific). Western blots were performed using the iBind Automated Western System (Thermo Fisher Scientific). For protein detection, we used the following primary antibodies: nucleocapsid protein (catalogue no. ab272852; Abcam); POP1 (catalogue no. 12029-1-AP; Proteintech); LARP1; CNBP; α-Tubulin (catalogue no. 2144; Cell Signaling Technology); β-Actin (catalogue no. sc-47778; Santa Cruz Biotechnology). We used the following secondary antibodies: IRDye 800CW goat anti-rabbit IgG (LI-COR); IRDye 800CW goat anti-mouse IgG (LI-COR). For the visualization of bands, we used the Odyssey Clx Infrared Imager System (LI-COR).

Core specific IgG antibodies were analyzed by ELISA as described previously with some modifications (20 (link)). In brief, Maxisorb 96 well plates (Nunc, Denmark) were coated overnight at 4°C with 0.5 µg of nucleocapsid protein (Abcam, The UK) dissolved in 100 µL carbonate coating buffer (CBC). Wells were washed with washing buffer (0.05% Tween 20 in PBS) and blocked to inhibit non-specific protein binding with blocking buffer (3% BSA in PBS). Mice sera were applied to wells and incubated for 2 h at 37°C. After washing steps, 100 µL of 1:7000 diluted HRP-conjugated anti- mice IgG (Sigma) was applied for 1 h at 37°C. TMB (tetramethylbenzidine) was added to wells to develop colorimetric reaction, and optical density (OD) was measured at 450nm with a plate reader after the reaction was stopped by adding 2N H2SO4. To determine anti-core specific IgG subtypes, IgG1 and IgG2a subclasses were measured using goat anti-mouse IgG1 and IgG2a secondary antibodies (Sigma) according to the same protocol and manufacturer’s instructions.