Cellulose membranes

Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 4–12% Criterion gels (BioRad, Hercules, California) with MOPS running buffer and transferred to cellulose membranes (GE Healthcare, Little Chalfont, United Kingdom) with the Criterion Blotter system (BioRad). The following antibodies were used: an anti-STrEP-Tag (#34850, Qiagen, Hilden, Germany), anti-LGP2 (NBP1-85348, Novus, Littleton, Colorado), anti-MDA5 (#5321, Cell Signaling, Danvers, Massachusetts and AT113, EnzoLifescience, New York, NY), anti-RIG-I (D14G6, Cell Signaling) or monoclonal anti-β-actin antibody (A5441, Sigma), IRF3 (#11904, Cell Signaling) or phosphor-IRF3 (ab76493, Abcam, Cambridge, UK,). HRP-coupled anti-mouse (NA9310V, GE Healthcare) or anti-rabbit (RPN4301, GE Healthcare) were used as secondary antibodies. Peroxidase activity was visualized with an ECL Plus Western Blotting Detection System (#RPN2132, GE Healthcare). MV intracellular staining was performed with mouse anti-N mAb (clone 25, [Giraudon and Wild, 1981 (link)]) and FITC coupled Goat Anti-mouse Ab (BD Biosciences, Franklin Lakes, New Jersey). For CHIKV, intracellular staining was performed with either FITC-conjugated anti-CHIK.E2 mAB 3E4 (Bréhin et al., 2008 (link)) or anti-dsRNA mAb (J2-1201, Scicons, Szirák, Hungary) followed by anti-mouse-APC antibody (A865, Invitrogen).

SDS-polyacrylamide gel electrophoresis was performed on 4–15% Mini-PROTEAN TGX Stain-Free gels (BioRad; #4568085) or NuPAGE 3–8% Tris-Acetate gels (Invitrogen, #EA03752BOX), then transferred to cellulose membranes (GE Healthcare, Little Chalfont, UK) with Trans-Blot^® SD Semi-Dry Transfer Cell. The following antibodies were used: anti-VP1 (1/500, Dako, 5D8/1), anti-eIF4G (1/1000, #2469S, Cell Signaling Technology, Danvers, MA, USA) and monoclonal anti-β-actin antibody (1/1000, #3700, Cell Signaling Technology). HRP-coupled anti-mouse (1/5000, NA9310V, GE Healthcare) and anti-rabbit (1/10,000, #7074S, Cell Signaling Technology) were used as secondary antibody. Peroxidase activity was visualized with an ECL Plus Western Blotting Detection System (#RPN2132, GE Healthcare).