Hrp linked anti rabbit or anti mouse antibody

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).

The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (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% skim milk (Wako), and incubated with specific antibodies in Can Get Signal immunoreaction enhancer solution (Toyobo) at 4°C overnight. Anti-TRBP (AbFrontier, Seoul, Korea), anti-AGO2 (Wako), anti-PKR (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho PKR (Abcam, Cambridge, UK), anti-FLAG (Cell Signaling, Danvers, MA, USA) and anti-α-tubulin antibodies (Abcam) were used. Anti-human RIG-I, anti-MDA5, anti-LGP2, and anti-Dicer antibodies were generated by immunizing rabbits with a synthetic peptide (8 (link),31 (link)). The membranes were 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) and visualized using the ImageQuant LAS 4000 Mini imager (GE Healthcare).