Epr8590 448

Viral proteins were detected using in-house produced rat polyclonal antibodies against NP protein (1:5000; [24 (link)]) and against NS1 protein [25 (link)]. Cellular proteins and their modifications were detected using commercially available antibodies as follows: rabbit polyclonal antibodies to H3K79me2 (D15E8), H3 (D1H2), and CHD1a (D8C2) (1:1000) from Cell Signaling (Danvers, MA, USA); rabbit polyclonal antibodies anti-TRIM25 (EPR7315) (ab167154), anti-RIG-I (DDX58) (ab45428), and anti-Ubiquitin UbK63 (linkage-specific K63) (EPR8590-448) (ab179434) from Abcam (Cambridge, United Kingdom); mouse monoclonal antibodies anti-MAVS (E-3 (sc-166583); 1:1000), and anti-β-tubulin (1:1000) from Santa Cruz (Dallas, TX, USA) and Sigma-Aldrich, respectively.

Proteins were separated by SDS-PAGE on 4–12% NuPAGE Bis-Tris gels (Life Technologies) and visualized with Oriole fluorescent gel stain (Bio-Rad) or Bio-Safe Coomassie Stain (Bio-Rad). For immunoblotting, the proteins were transferred to polyvinylidene fluoride membrane (GE Healthcare) on an XCell II Blot Module (Life Technologies). After verifying transfer by Ponceau S staining, we performed immunoblotting with the following antibodies: mouse monoclonal antibody against Ub (P4D1, horseradish peroxidase (HRP)-conjugated; used at 1:500 for immunoblotting; Santa Cruz Biotechnology); mouse monoclonal antibody against the FLAG-tag (M2, HRP-conjugated; 1:1000; Sigma-Aldrich); mouse monoclonal antibody against EGFR (6F1; 1:1000; MBL); rabbit monoclonal antibody against K48-linked (EP8589; 1:1000; Abcam); and rabbit monoclonal antibody against K63-linked (EPR8590-448; 1:1000; Abcam). HRP-conjugated goat anti-mouse Ig (1:10 000), used as a secondary antibody, was purchased from Jackson ImmunoResearch Laboratories. Immunoblots were developed using ECL Prim Western Blotting Detection Reagent (GE Healthcare), and analyzed on an ImageQuant LAS4000 (GE Healthcare). Uncropped blot images are shown in Supplementary Fig. 9.