Fig. 1.

Expression of recombinant IBV S1 and H5N1 HA proteins. (A) Schematic representation of the S1 and HA expression cassettes. The S1 or HA ectodomain-encoding sequences were cloned in frame with DNA sequences coding for a signal sequence (ss), the GCN4 isoleucine zipper trimerization motif, and Strep-tag II (ST) under the control of a cytomegalovirus (CMV) promoter. (B) S1 proteins expressed in HEK293T cells and purified from the culture medium were analyzed by SDS-PAGE followed by Western blotting using Strep-Tactin. Where indicated, the samples were treated with PNGase F prior to electrophoresis. (C) Fetuin binding was analyzed by a solid-phase binding assay as described in Materials and Methods. The graph shows the results of an independent experiment performed in triplicate. (D) Spike and HA histochemistry was performed by incubating chicken trachea and lung tissues with Strep-Tactin-precomplexed M41 S1 (5 μg) or H5N1 HA (0.1 μg) as described in Materials and Methods. Binding is indicated by arrowheads.