FIG. 2.

Localization of the FHL-1 and FH binding sites in Fba. (A) Coomassie blue-stained SDS-PAGE gel of the recombinant Fba proteins used for this study. The name of each Fba derivative is listed above the gel. MW, molecular mass standards. Numbers to the left of the figure indicate the sizes of each protein standard, in kilodaltons. (B) FH binding by Fba derivatives. The same proteins shown in panel A were transferred to nitrocellulose, blocked, and incubated successively with 10 μg of FH per ml, goat anti-human FH antibody, and an alkaline phosphatase-labeled secondary antibody. No bands were detectable on a duplicate blot incubated with only the primary and secondary antibodies (not shown). At least two independent experiments were performed with each Fba derivative. (C) FHL-1 binding by Fba derivatives. Binding was performed as described for panel B except that the membrane was incubated with FHL-1. (D) FH binding to immobilized Fba. The recombinant Fba derivatives were adsorbed onto microtiter plates and incubated with various concentrations of FH. Binding was detected with FH antiserum and a labeled secondary antibody. Data represent the means from a representative experiment, wherein each assay was performed in triplicate. Each recombinant protein was tested in at least two independent experiments. (E) FHL-1 binding by Fba derivatives. Binding assays were performed as described for panel D except that FHL-1 was substituted for FH. A₄₀₅, absorbance at 405 nm.