Western Blot Analysis of Protein Expression

The samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membrane was blocked for 1 h in Tris-buffered saline-Triton X-100 or Tween 20 (TBS-T; 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.2% Triton X-100, or 0.1% Tween) supplemented with 5% Difco skim milk (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and incubated with specific antibodies in Can Get Signal immunoreaction enhancer solution (TOYOBO, Osaka, Japan) at 4 °C overnight. Anti-Myc or anti-FLAG (Cell Signaling, Danvers, MA, USA), anti-AGO2 (Wako, Osaka, Japan), and anti-α-tubulin antibodies (ICN/CAPPEL Biomedicals, Santa Ana, CA, USA) were used. Anti-human RIG-I, MDA5, and LGP2 antibodies were generated by immunizing rabbits with a synthetic peptide [41 (link)]. The membrane was washed three times with TBS-T, and reacted with HRP-linked anti-rabbit or anti-mouse antibody (GE Healthcare, Chicago, IL, USA) at room temperature for 1 h. After being washed three times with TBS-T, the membrane was incubated with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) and visualized with the LAS3000 system (Fujifilm, Osaka, Japan).