Western lightning ultra substrate

Samples were separated by SDS-polyacrylamide (10-16.5%) gel electrophoresis and electroblotted onto a membrane filter (Immobilon-P; MilliporeSigma, Burlington, MA, USA). Membranes were blocked with Blocking One (Nacalai Tesque) for 30 min, followed by incubation at 20–25°C with rabbit polyclonal antibodies against FLAG (MBL), V5 (MBL), Myc (MBL), HA (MBL), MAVS (D5A9E) (#24930, Cell Signaling Technology, Danvers, MA, USA), TRIM25 (#13773, Cell Signaling Technology) or a mouse monoclonal antibody against RIG-I (D-12) (sc-376845, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h. Membranes were then incubated at 20–25°C for 30 min with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (GE Healthcare, Chicago, IL, USA). Protein bands were visualized using enhanced chemiluminescence Western Lightning Ultra Substrate (PerkinElmer, Waltham, MA, USA) and FUSION-Solo S Imaging System (Vilber Lourmat Sté, Collégien, France).

Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Two sample loading strategies were employed: 10 μg protein was loaded for fractions with sufficient protein (usually F5&6, F7, and F8&9), while 30 μl of the sample was loaded for other fractions with less than 10 µg per 30 μl. All samples were prepared in 4× Laemmli sample buffer (Bio-Rad, Benicia, CA, USA) containing β-mercaptoethanol and heated at 95°C for 15 min. Samples were separated on 4–15% Mini-PROTEAN TGX precast Gels (Bio-Rad) on a Mini PROTEAN Tetra Cell system (Bio-Rad) and transferred to an Immobilon-P PVDF membrane (Millipore) following the manufacturer’s guidelines. The membrane was then blocked with 5% BSA for 1.5 h at RT and incubated overnight at 4°C with the primary antibodies. After incubation with primary antibodies, the membranes were washed with 0.05% Tween in phosphate-buffered saline (PBST) six times for 5 min each. Secondary antibody staining was conducted at RT for 1 h. After washing with PBST, the membranes were detected using Western Lightning Ultra substrate (PerkinElmer). Images were taken by using a UVP ChemStudio PLUS Touch system (Analytic Jena). All the antibodies used in this study are listed in Supplementary Table .