Odyssey clx infrared imager system

Protein samples were denatured at 95 °C and resolved by 12% SDS-PAGE at 30 mA for 2 h. After transfer using Trans-Blot (Bio-Rad), nitrocellulose membranes were developed using the following primary antibodies: anti-His-tag (ab18184, dilution 1:1000), anti-DDDDK (ab49763, dilution 1:3000), anti-ALFA (FluoTag^®-X2 anti-ALFA AlexaFluor 647, dilution 1:1000), anti-ZC3HAV1 (Proteintech 16820-1-AP, dilution 1:3000), anti-RPL4 (Proteintech 67028-1-Ig, dilution 1:10000), anti-RPS6 (Proteintech 14823-1-AP, dilution 1:500), anti-RYDEN (SHFL; Proteintech 27865-1-AP, dilution 1:1000). The following secondary antibodies were used: IRDye^® 800CW Goat anti-rabbit (dilution 1:25000) and IRDye^® 680RD Donkey anti-Mouse (dilution 1:15000; both LI-COR). Bands were visualized using an Odyssey Clx infrared imager system (LI-COR) or a Typhoon7000 (GE Healthcare).

For western blot analysis, 2x10⁵ cells were rinsed with PBS and lysed in 80 μl Laemmli buffer (2% SDS, 10% Glycerol, 60 mM Tris, 0.01% (w/v) bromphenol blue, 50 mM DTT) and lysates were sheared by passing through a syringe. Proteins were separated by SDS PAGE in NuPAGE 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific) or 3-8% Tris-Acetate Protein Gels (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane using the iBlot dry blotting system (Thermo Fisher Scientific). Membranes were incubated with anti-SND1 (Proteintech, 60265-1-Ig), anti-Flag (Sigma, F3165), anti-HA (Proteintech, 66006-2-Ig), anti-V5 (Abcam, ab9116) or anti-Actin (Sigma Aldrich, A2103) antibodies using the iBind Automated Western System (Thermo Fisher Scientific) and imaged in the Odyssey Clx Infrared Imager System (LI-COR).

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).