Treatment with PNGaseF allowed for the removal of N-linked glycosylations, as shown by the reduction of apparent molecular weight of HA0, HA1, and HA2 species in Western blot analysis for HA proteins expressed in cells transfected by these H5-VN HA DNA vaccines ( Figure S1 ). The molecular weight reduction patterns for HA0, HA1, and HA2 antigens were the same between cells transfected by H5-VN.wt and H5-VN.tPA DNA vaccines, suggesting that HA antigens expressed by these two HA DNA vaccines were similarly glycosylated. A smaller molecular weight HA2 antigen was observed in cells transfected with the H5-VN.dTM DNA vaccine, presumably due to the truncated size of HA2 domain in this particular HA insert design; however, PNGaseF treatment also led to a proportional reduction of molecular weight for truncated HA2 protein in both supernatant and cell lysate preparations. The only unique finding is that the cell-associated HA antigens in H5-VN.dTM transfected cells showed a high level of heterogeneity and a small portion of the HA1 proteins was not fully deglycosylated by PNGaseF treatment, reflecting the continued presence of different forms of glycosylated HA proteins in H5-VN.dTM transfected cells. Otherwise, the overall glycosylation pattern, as probed by deglycosylation treatment, was very similar among HA antigens produced by three different types of H5 HA DNA vaccines.