Equal amounts of protein from total cell lysates or equal volumes of H5pp concentrated by ultracentrifugation were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto Hybond-P polyvinylidene difluoride (PVDF) membranes (Invitrogen) that were blocked with 5% milk for 30 min at room temperature. H5-HA was detected by incubation for 2 hr at room temperature with a mouse monoclonal anti-FLAG M2 antibody (Sigma, St.Louis, MO, USA; 1:1000 dilution) conjugated with horse radish peroxidase (HRP) (Sigma). The core protein in the pseudotyped particles was detected using an anti-p24 antibody (Abcam, Cambridge, UK) for 1 hr at room temperature at a 1:1000 dilution, followed by an additional 1 hr incubation with a goat-anti-mouse secondary antibody conjugated with HRP (ZymedH, Invitrogen) at a 1:5000 dilution. The levels of cyclophilin B (detected with a rabbit anti-cyclophilin B antibody from Abcam, 1:5000 dilution) or GAPDH (detected with a mouse anti-GAPDH antibody from Abcam, 1:10000 dilution) were measured on the same blots to verify that equal amount of samples had been transferred. Proteins were visualized by chemiluminescence using ECL Western blot detection reagents (Invitrogen). The relative electrophoretic mobility was estimated using NovexH Sharp Pre-stained Protein Standards (Invitrogen).