At 72 hr post-transfection, the cells transfected with HAexpressing plasmids were harvested, pelleted, and lyzed in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, pH 7.4) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN). Aliquots of cell lysates (50 mg) or virus lysates were blotted after 10% SDS-PAGE onto nitrocellulose membranes (Pall, Port Washington, NY). The membranes were blocked with 5% non-fat milk and then incubated with the primary antibodies indicated in the figures at 4uC overnight. This was followed by incubation with the goat anti-mouse IRDyeH Fluor 800-labeled IgG secondary antibody (1:10, 000) (Li-Cor, Lincoln, NE). After washing, the membranes were scanned by the OdysseyH Infrared Imaging System (Li-Cor) and analyzed with Odyssey software. The molecular sizes of the developed proteins were determined by comparison with the pre-stained protein markers (Fermentas, Maryland, CA).