Anti ha tag antibody

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 Anti-VP1 polyclonal antibody was prepared by our laboratory (unpublished data).

Cells were plated on 0.1% gelatin (wt/vol) coated glass coverslips and fixed using 4% paraformaldehyde (wt/vol) in PBS. Fixed cells were permeabilized with 0.5% Triton X-100 (vol/vol) in PBS and blocked with coverslip block buffer (4% Fish gelatin [wt/vol], 5.0% goat serum [vol/vol], and 1.2% BSA [wt/vol] in PBS). Cells were then incubated with an anti HA-tag antibody (1:10,000; BioLegend) followed by an Alexa 488-conjugated anti-mouse secondary antibody (1:500; Thermo Fisher Scientific) diluted in coverslip block buffer in a humid chamber. Actin red 555 reagent (Thermo Fisher Scientific) was added to the secondary antibody mix to stain actin. DNA was stained using Hoechst. Cells were mounted on glass slides using FluorSave (MilliporeSigma). Z-stack confocal images were acquired with a Nikon A1R confocal microscope using the NIS-Elements software. Maximum intensity z-projections were generated using Image J (NIH) and images were assembled using Photoshop and Illustrator (Adobe). Quantification of the nuclear versus total HA-USP27X signal was performed using the adjust threshold and analyze particles tools of Image J (NIH).