(A) SDS-PAGE of LOS isolated from wild-type NTHi 2019 and NTHi 2019siaT. Lanes 1 to 4, LOS isolated from wild-type NTHi 2019; lanes 5 to 8, LOS isolated from the mutant NTHi 2019siaT. Bacteria were grown on BHI in the absence (lanes 1, 2, 5, and 6) or presence (lanes 3, 4, 7, and 8) of Neu5Ac. LOS samples in lanes 2, 4, 6, and 8 were treated with neuraminidase prior to loading. LOS from N. gonorrhoeae strain PID2 was used as a molecular weight standard. The LOS was visualized using silver staining. (B) Western blot of LOS probed with 3F11. Lanes 1, 3, 5, and 7, LOS isolated from wild-type NTHi 2019; lanes 2, 4, 6, and 8, LOS isolated from the mutant NTHi 2019siaT. Bacteria were grown on BHI in the absence (lanes 1 to 4) or presence (lanes 5 to 8) of Neu5Ac. LOS samples in lanes 3, 4, 7, and 8 were treated with neuraminidase prior to loading. LOS from N. gonorrhoeae strain PID2 was used as a molecular weight standard. LOS was probed with monoclonal antibody 3F11, which recognizes a terminal N-acetyllactosamine structure. Antibody binding was detected using peroxidase-labeled goat anti-mouse IgM and a chemiluminescent substrate.