FIG. 5.

Anti-Ecgp antibody recognizes HBMEC surface proteins. (A) Biotinylated HBMEC proteins (0.2 mg) were preincubated with 1 ml of agarose to preclear the nonspecific binding proteins. The supernatant was incubated with 1 μg of anti-Ecgp antibody overnight at 4°C, followed by incubation with protein A-agarose. The immunocomplexes were released by Laemmli buffer, separated by SDS-PAGE, and probed with streptavidin-peroxidase. An unrelated antibody was used as a control (Cont-Ab). (B) HBMEC and HUVEC membrane proteins were subjected to Ecgp-Ab affinity chromatography as described in Materials and Methods. The bound proteins were eluted and subjected to immunoblotting with Ecgp-Ab.