Ab226819

(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).

Twelve microliters of supernatant or cell lysate were separated on a 4–12% Mini-Protean TGX gel (Bio-Rad) at 100 V for 1 h. Proteins were transferred onto a 0.2 μm nitrocellulose membranes (Li-Cor Biotechnology) at 100 V for 1 h. The membranes were blocked in 5% non-fat dry milk in PBS containing 0.2% Tween-20 for 1 h followed by incubation in PBS containing 5% BSA, 0.2% Tween-20, and either 1:1,000 rabbit polyclonal anti-Caspase-1 p20 (47 (link)), anti-IL-1β (70 (link)), anti-HMGB1 (abcam; ab18256), anti-PKR (phospho T446) (abcam; ab32036), anti-PKR (abcam; ab226819), anti-beta Actin antibody (abcam; ab8229), Anti-GFP antibody (abcam; ab6556), or 1:500 anti-Asc (Tyr-144) phospho-specific antibody (ECM Bioscience; AP5631) with rotation overnight at 4°C. The following day, donkey anti-rabbit IRDye 800CW and donkey anti-goat IRDye 680RD secondary antibodies (Li-Cor Biotechnology) were applied at a dilution of 1:15,000 with rocking for 1 h at room temperature. Membranes were imaged on a Li-Cor Odyssey CLx machine with auto exposure and high-quality setting.