Mouse monoclonal anti hemagglutinin ha

HEK293 cells transfected with plasmids encoding Lphn3 receptor variants were harvested 48 h post-transfection, solubilized in Laemmli sample buffer and resolved by electrophoresis on 10% SDS-PAGE gels before being transferred onto nitrocellulose membranes (Merck-Millipore, Burlington, MA, USA). A blocking step was conducted using 3% BSA in 1× TBST (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 2 h at room temperature prior to adding primary antibodies: mouse monoclonal anti-hemagglutinin (HA) (1:1000 ratio in blocking solution; BioLegend, San Diego, MA, USA; 901513), rabbit polyclonal anti-Flag (1:1000 ratio in blocking solution; Sigma-Aldrich, F7425) and mouse monoclonal V9 anti-vimentin antibody (1:250 ratio in blocking solution; Santa Cruz Biotechnology, Dallas, TX, USA; sc-6260) followed by anti-rabbit IRDye680RD and anti-mouse IRDye800CW as secondary antibodies (1:15,000 ratio in blocking solution; LI-COR, 926-68071 and 926-32212, respectively). Finally, membranes were scanned using 700 nm and 800 nm channels from LI-COR Biosciences Odyssey Fc imaging system to quantify fluorescent signals.

The full-length porcine NME1 cDNA was amplified and inserted into pcDNA^TM3.1/myc-His(-)A vector (Invitrogen) to generate a plasmid expressing Myc-tagged NME1 (Myc-NME1). A series of plasmids expressing Flag-tagged viral proteins were constructed as described previously⁵² . HA-tagged p53 plasmid (HA-p53) was constructed by inserting full-length p53 cDNA into pCAGGs vector (including a C-terminal HA tag). Flag-p53, p53-Luc reporter plasmids and control plasmid Renilla luciferase pRL-TK were kindly provided by Zhiyong Ma (Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China)^{53 (link)}. All the expressing plasmids used in this study were analyzed and verified by DNA sequencing.
Commercial antibodies included: mouse monoclonal anti-Myc, monoclonal anti-Flag, rabbit polyclonal anti-NME1, and mouse monoclonal anti-β-actin (Santa Cruz Biotechnology); mouse monoclonal anti-hemagglutinin (HA) (BioLegend); mouse monoclonal anti-Flag (Sigma); rabbit polyclonal anti-eukaryotic translation initiation factor 4 gamma (eIF4G) (Abcam); rabbit polyclonal anti-LC3B (Sigma); rabbit monoclonal anti-ATG12 (Cell signaling technology); rabbit polyclonal anti-VP1 antibody was prepared by our laboratory as described previously^{48 (link)}.