Fig 4.

Western blots used for the determination of relative viral protein contents of samples reported in Tables 3 and 4. (A) Eggs were inoculated with 100 PFU of either PR8(Ud-HA,NA) or PR8(Ud-HA,NA,PB1). Allantoic fluid was clarified by ultracentrifugation, and the virus was pelleted at 31,000 × g and then resuspended in PBS. Tris-glycine gels (4 to 20%) were loaded with 20,000 HAU of virus sample and separated by SDS-PAGE under reducing conditions. Proteins were transferred to a PVDF membrane and probed with either anti-NP or anti-HA2 monoclonal antibodies. The relative protein contents were determined by densitometry of stained bands analyzed using the ImageQuant TL software and are presented in Table 3. (B) Monovalent split virus vaccine preparations were deglycosylated, and samples containing 21 μg of viral protein, as determined by Lowry assay, were separated on 10% bis-Tris gels under reducing conditions in the presence of NuPAGE antioxidant. Proteins were transferred to a PVDF membrane and probed with either anti-NP or anti-HA2 monoclonal antibodies. The relative protein contents were determined by densitometry of stained bands analyzed using the ImageQuant TL software and are presented in Table 4.