Protein Extraction and Western Blot Analysis

To analyze gene knockdown or knockout, cells were lysed with NETN extract buffer containing 100mM NaCl, 20mM Tris-Cl (pH 8), 0.5mM EDTA, 0.5% Nonidet P-40, protease inhibitor cocktail (cOmplete, Roche) and phosphatase inhibitor cocktail (phosSTOP, Roche) for 30 min on ice. Lysates were cleared by centrifugation at 12,000 rpm for 10 minutes at 4°C. Proteins were visualized on SDS-PAGE. Western blotting was performed according to the standard protocols. The following antibodies were used at the following concentrations: anti-MAVS at a 1:1,000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA), HA at a 1:1000 dilution (Biolegend), anti-PKCα, anti-PKC-δ, anti-PKCµ, anti-PKCζ were each used at a 1:1,000 dilution (Cell Signaling, Danvers, MA), and anti-β-actin antibody at a 1:10,000 dilution (Sigma-Aldrich, Saint Louis, MO). Secondary anti-rabbit and secondary anti-mouse antibodies were used at a 1:10,00 dilution (Jackson ImmunoResearch, #111-035-046 and 115-035-146, respectively). To analyze cytoplasmic and nuclear fractions, samples were prepared as previously described (28 (link)) and according to manufacturer instructions using the Qproteome cell compartment kit (Qiagen).