Western Blot Analysis of RIG-I, PKR, and MAVS

(Knockout) cells were grown in a 6-well plate until confluent, washed twice with cold PBS, and lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with phosphatase inhibitors (PhosSTOP EASYpack; Roche) and protease inhibitors (EASYpack protease inhibitor cocktail; Roche). After 5 min of incubation at 4°C, cells were scraped, transferred into Eppendorf tubes, and incubated in a tube revolver (Thermo Fisher Scientific) for 1 h at 4°C. Samples were centrifuged for 15 min at 15,000 × g at 4°C. The supernatants were diluted in NuPAGE LDS sample buffer (Supplier: Thermo Fisher Scientific) (4×) and analyzed by Western blot analysis as described previously (45 (link)). Monoclonal antibodies targeting RIG-I (AG-20B-0009-C100; Bio-Connect), PKR (ab226819; Abcam), and MAVS (ab89825; Abcam) were used according to the manufacturer’s instructions. Secondary polyclonal rabbit anti-mouse IgG-horseradish peroxidase (HRP) (P0260; Agilent) and polyclonal swine anti-rabbit IgG-HRP (P0217; Agilent) antibodies were used at a 1:1,000 dilution. Imaging was performed using an Amersham imager 600 (GE Healthcare).