Anti flag monoclonal antibody

HA-tagged Nrd were co-transfected either with FLAG-tagged zebrafish Pin1 or FLAG-vector control. Cell lysates were collected in mammalian cell lysis buffer (50 mM Hepes pH 7.4; 10% glycerol; 1% Triton X-100; 100 mM NaCl and 0.5 mM MgCl₂) supplemented with protease and phosphatase inhibitors. Supernatants were incubated with FLAG-M2 beads (Sigma) for 1.5 h at 4°C. Bound proteins were eluted by 2 × SDS loading dye. The samples were analyzed on SDS-PAGE and Western blotting with polyclonal anti-HA antibody (Zymed) and monoclonal anti-FLAG antibody (Santa Cruz Biotechnology).

The cDNA of porcine RIG-I were amplified from PK-15 cells and cloned into pcDNA3.1/myc-His(−)A vector (Invitrogen) to yield the Myc-tagged expression construct (Myc-RIG-I). Each of FMDV full-length viral cDNA was inserted into p3xFLAG-CMV-7.1 vector (Sigma-Aldrich) to construct plasmids expressing Flag-tagged viral proteins. A series of Flag-tagged truncated 2B constructs were generated by site-directed mutagenesis PCR. All constructed plasmids were analyzed and verified by DNA sequencing. The IFN-β promoter luciferase reporter plasmids and various hemagglutinin (HA)-tagged plasmids used in this study were kindly provided by Hongbing Shu (Wuhan University, China) (25 (link)). The commercial antibodies used in this study include: anti-Myc monoclonal antibody (Santa Cruz Biotechnology), anti-Flag monoclonal antibody (Santa Cruz Biotechnology), anti-Flag polyclonal antibody (Sigma-Aldrich), anti-RIG-I polyclonal antibody (Abcam), anti-HA tag antibody (BioLegend), anti-eukaryotic translation initiation factor 4 gamma (eIF4G) polyclonal antibody (Abcam), and anti-β-actin monoclonal antibody (Santa Cruz Biotechnology). Anti-VP1 polyclonal antibody was prepared by our laboratory (unpublished data).