Immunostaining of recombinant H5-HA (lane 1, 85 kDa), H5N1 vaccine virus lysate (lane 2) and pseudovirus 1194 lysate (lane 3) by polyclonal anti-HA5 Ab, mAb C, 3C4, or 6B8. M indicates lanes for protein size markers. The anti-HA polyclonal antibody recognizes HA0 or HA2 (25-35 kDa) better than HA1 (50-60 kDa; left panel) but mAb C, 3C4, and 6B8 recognize intact hemagglutinin HA0 only (right 3 panels). The H5N1 vaccine virus has the polybasic site removed, so it is not recognized by mAb C, 3C4, or 6B8 (lane 2 in each of right 3 panels). Based on densitometry analysis, it is estimated that the ratio of HA0 (full length) to HA1+HA2 (proteolytic products) are about 1:3 on recombinant HA or pseudovirus 1194 lysate (left panel, lane 1 and 3).