Trim25

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).

Primary antibodies against the following proteins were obtained from Cell Signaling Technology: BiP (#3183, 1:1000), JNK (#9252, 1:1000), Phospho-SAPK/JNK (Thr183/Tyr185) (#9251, 1:1000), eIF2α (#9722, 1:1000), Phospho-eIF2α (Ser51) (#9721, 1:1000), cleaved caspase-3 (Asp175) (#9661, 1:1000), KEAP1 (P586) (#4678, 1:1000); from Sigma: Flag M2 (F3165, 1:2000), tubulin (T6074, 1:2000); from MBL: GFP (M048-3, 1:1000), His (D291-3, 1: 3000); from Thermo Fisher Scientific: HA (SG77) (71-5500, 1:1000); from Novus Biologicals: TRIM25 (NBP2-20710, 1:1000); from Santa Cruz Biotechnology: Nrf2 (A-10) (sc-365949, 1:1000); from Proteintech: KEAP1 (10503-2-AP, 1:200) and Nrf2 (16396-1-AP, 1:200); from Abclonal: TRIM25 (A12938, 1:200); from Cell Signaling Technology: anti-rabbit IgG, HRP-linked Antibody (7074, 1:3000) and anti-mouse IgG, HRP-linked Antibody (7076, 1:3000);from Invitrogen: Goat anti-Mouse IgG, Alexa Fluor 488 (A-11001, 1:500) and Donkey anti-Mouse IgG, Alexa Fluor 568 (A-10037, 1:500).

Proteins were extracted from the cell using RIPA lysis buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail (MCE, Shanghai, China), then transferred onto PVDF membrane to perform SDS-PAGE (Millipore, Billerica, MA, USA). Next, the PVDF membrane was incubated in phosphate buffer saline (PBST) containing 0.05% Tween-20 and 5% non-fat milk for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C, and secondary antibodies (1:10,000, proteintech) for 1 h at room temperature. Blots were developed using ECL (Tanon, Shanghai, China). The primary antibodies were as follows: TRIM25 (Cell Signaling technology, 1:1000 dilution, #13773), DDX5 (Cell Signaling technology (Danvers, MA, USA), 1:1000 dilution, #4387), YBX1 (ABclonal (Woburn, MA, USA), 1:2000 dilution, A7704), DHX9 (Abcam (Cambridge, UK), 1:1000 dilution, ab26271). Loading control antibodies are as follows: GAPDH (ABclonal, 1:2000 dilution, A19056), Vinculin (SantaCruz (Santa Cruz, CA, USA),1:1000 dilution, V284), H3 (Cell Signaling technology, 1:1000 dilution, #4499).