To analyze the N-linked glycosylation of H5 HA antigens expressed by various forms of H5 HA DNA vaccines, the HA antigens expressed from 293T cells [45, 46] were treated with PNGaseF (New England BioLab, Beverly, MA) [47, 48, 49] . Briefly, the HA proteins were first denatured at 100uC for 10 min in glycoprotein denaturing buffer and then chilled on ice. Following addition of G7 reaction buffer, the deglycosylation enzyme cocktail was added and incubated reaction at 37uC for 4 hours. Either mock-treated or deglycosylated HA samples were subjected to SDS-PAGE and Western blot analysis was performed as described above.