Western Blot Analysis of Innate Immune Proteins

Cell lysates were prepared using M-PER Mammalian Protein Extraction Reagent with a cocktail of protease and phosphatase inhibitors (Cell Signaling). The protein samples were separated on an NU-PAGE Bolt Bis-Tris Plus Gels (Invitrogen), transferred onto nitrocellulose membranes (Bio-Rad), blocked using Intercept (TBS) Blocking Buffer (Li-Cor Biosciences), and then incubated overnight with different antibodies (Strange et al., 2019 (link)). Specific primary monoclonal antibodies used were rabbit anti-human against RIG-I (Cell Signaling Cat. No: 3743S), MDA5 (Invitrogen Cat. No: 700360), MXA (Santa Cruz Biotechnology Cat. No: sc-271024), IRF3 and phospho-IRF3 (Cell Signaling Cat. No: 11904S and Cat. No: 4947S), STAT1 (Cell Signaling Cat. No: 9172S), phospho-STAT1 (Cell Signaling Cat. No: 9167L), STAT2 (Cell Signaling Cat. No: 72604S), phospho-STAT2 (Cell Signaling Cat. No 4441) and mouse anti-human β-actin (Sigma Cat. No. A2228-200UL), all at 1:1,000 dilution. Secondary antibodies (1:10,000 dilution) were conjugated with IRDye 800 and IRDye 680 (Li-Cor Biosciences), and blots were scanned using an Odyssey infrared imager.