Ecsit

Cultured cells were washed twice with ice-cold PBS and lysed with a buffer containing 20 mM Tris-HCL, 150 mM NaCl, 1% Triton X-100, and protease inhibitor mixture, and phosphatase inhibitor cocktail (P8340, Sigma-Aldrich) on ice for 30 minutes. The cell lysates were centrifuged at 13,200 rpm for 15 min at 4°C, and the supernatants were collected in new 1.5 ml microcentrifuge tube. The protein concentration of cell lysates was determined using a DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). The extracted protein (30-40 μg per sample) was subjected to 10%–12% SDS-PAGE gels and transferred electrophoretically onto polyvinyl difluoride membranes (MilliporeSigma). The membranes were blocked in 5% fat-free milk/Tris-buffered saline for 90 min and incubated with each primary antibody overnight, followed by secondary antibodies conjugated with horseradish peroxidase. The following monoclonal antibodies were used: ECSIT (Abcam, Cambridge, UK; ab21288; 1:1000), TRAF6 (Santa Cruz Biotechnology, Santa Cruz, CA; sc-8409; 1:1000), VDAC (Cell Signaling, MA, USA; D73D12; 1:1000), β-actin (Sigma-Aldrich; A5316; 1:5000). Blots were stripped and reprobed with anti–β-actin or anti–VDAC antibodies. Bound antibodies were detected with ECL reagents (MilliporeSigma) and were imaged and quantified with a VersaDoc imaging system (Bio-Rad).

Cell lysates were quantified by Bradford assay. According to the detected protein size, SDS-PAGE gels at various percentages (8–13.5%) were used to separate proteins. For immunoblotting and immunofluorescence, antibodies against β-actin (Bioss, cat. #BS-0061R, Woburn, MA, USA), GAPDH (Millipore; cat. #MAB374, Burlington, MA, USA or Santa Cruz; cat. #sc-47724, Dallas, TX, USA), VDAC1 (Santa Cruz, cat. #sc-8828), MT-CO2 (Life Technologies, cat. #A6404, Carlsbad, CA, USA), p65 (Santa Cruz, cat. #sc-372 or sc-8008), phosphor-IRF3 (Cell Signaling, cat. #4947S, Danvers, MA, USA), ECSIT (Abcam, cat. #ab21288), MAVS (Santa Cruz, cat. #sc-166583), RIG-I (Santa Cruz, cat. #sc-376845), NDV HN (Santa Cruz, cat. #sc-53562), influenza A virus NP (Abcam, cat. #ab128193), TOM20 (Santa cruz, cat. #sc-17764) and HDAC1 (Santa Cruz, cat. #sc-6298) were used. For immunofluorescence staining, cells were seeded on cover glass that had been coated with poly-D-lysine. Then, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 1% BSA. An Alexa Fluor 488-conjugated secondary antibody (Invitrogen, cat. #A11017) was used in this study. To stain mitochondria, cells were incubated with 100–400 nM MitoTracker Deep Red (Invitrogen, cat. #M22426) in serum-free DMEM for 30 min. DAPI solution (Invitrogen, cat. #R37606) was used to visualize nuclei.