Transient expression of the HA antigens from various HA DNA vaccine constructs were verified by Western blot analysis. HA DNA vaccine constructs were first transfected into the human embryonic kidney 293T cells using the calcium phosphate precipitation method. Briefly, 2610 6 293T cells at 50% confluence in a 60 mm dish were transfected with 10 mg of plasmid DNA, and a total of 3 ml supernatant and 100 ml of cell lysate were harvested 72 hours later. Equal amounts of each transiently expressed HA antigen (10 ng of protein in 10 ml) were loaded for the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under denatured conditions, then transferred onto PVDF membranes (Bio-Rad, Hercules, CA). After being blocked overnight at 4uC in blocking buffer (0.2% I-block, 0.1% Tween-20 in 16PBS), the membranes were incubated with a 1:500 dilution of rabbit sera immunized with HA DNA vaccines for 30 min followed by washes. Then, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG at 1:5000 dilution for 30 min. Following washes, the signals were detected using a chemiluminescence-based Western-Light Kit (Tropix, Bedford, MA).