Anti hemagglutinin ha antibody

A MAb against human ROBO1 was generated as previously described [14 (link),15 (link)]. Briefly, human ROBO1 cDNA was polymerase chain reaction (PCR)-amplified from Alexander cells and inserted into the pBlueBac 4.5-TOPO vector. The recombinant baculovirus expressing ROBO1 was immunized directly into gp64 transgenic mice. A positive hybridoma clone, B5209B, was selected by the reactivity to the ROBO1 stable cell line, by flow cytometry. An anti-hemagglutinin (HA) antibody was purchased from Sigma (St. Louis, MO, USA). MAb B5209B was purified by ammonium sulphate precipitation from the ascitic fluid of nude mice, to which the hybridoma cells were implanted intraperitoneally. To raise a MAb, which recognizes cell surface ROBO1, gp64 transgenic mice were immunized subcutaneously with 1 mg of ROBO1-expressing budded baculovirus with pertussis toxin adjuvant, as previously described [15 (link)].

Anti-DDX23, DDX3 antibodies were purchased from Abcam (Cambridge, MA, USA). The Anti-FLAG antibody was purchased from Proteintech (Chicago, IL, USA). Anti-hemagglutinin (HA) antibody, secondary antibodies conjugated with either horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC), and the chloroquine phosphate inhibitor was purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-actin antibody was purchased from Santa Cruz Biotechnology (CA, USA). The inhibitor Z-VAD(OMe)-FMK was purchased from Cell Signaling Technology (Danvers, MA, USA). XhoI and proteinase K were purchased from New England Biolabs (NEB, Ipswich, MA) Polyclonal pig antiserum directed against FMDV was prepared in our laboratory. MG-132 was purchased from Selleck Chemicals (Houston, TX, USA).

Localization experiments were performed using IFAs and laser scanning confocal microscopy. Briefly, MDCK cells grown on glass coverslips were transiently transfected with the appropriate tetherin expression constructs by employing Lipofectamine^® 3000. At 24 h after transfection, the cells were infected with CIV H3N2 at an MOI of 0.1, as described previously. At 48 h after infection, the cells were fixed with 4% paraformaldehyde-PBS. The cells were incubated with an anti-hemagglutinin (HA) antibody (Sigma, Saint Louis, MO, USA) to detect the HA protein and with anti-FLAG antibody (Sigma, Saint Louis, MO, USA) to detect canine tetherin and then they were stained using secondary antibodies conjugated with fluorescein isothiocyanate (FITC) and cyanine-3 (Cy3). After mounting on slides using antifade 4′,6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma), the cells were visualized with a Leica DM-IRE2 confocal microscope.