The samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). 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), and incubated with specific antibodies in Can Get Signal immunoreaction enhancer solution (TOYOBO) at 4°C overnight. Anti-TRBP (AbFrontier), anti-FLAG or anti-Myc (Cell Signaling), and anti-α-tubulin antibodies (ICN/CAPPEL Biomedicals) were used. Anti-human RIG-I, MDA5, LGP2 and Dicer antibodies were generated by immunizing rabbits with a synthetic peptide (4 (link),35 (link)). The membranes were washed three times with TBS-T and reacted with HRP-linked anti-rabbit or anti-mouse antibody (GE Healthcare) 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) and visualized with ImageQuant LAS4000 mini (GE Healthcare).