FIG 2 .

Binding of CARDS toxin to recombinant AnxA2. (A) Binding of CARDS toxin to AnxA2 by ligand blotting. GST-AnxA2 or BSA (2 µg each) was separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and incubated with CARDS toxin (7 µg/ml) for 2 h. CARDS toxin binding was detected by incubation with rabbit polyclonal anti-CARDS toxin antibody followed by incubation with goat anti-rabbit IgG and visualization with ECL. Lane 1, AnxA2; lane 2, BSA. (B) Dose-dependent binding of CARDS toxin to AnxA2. Microtiter wells were coated with 100 ng AnxA2, and increasing concentrations of CARDS toxin or BSA were added to individual wells for 1 h at room temperature. Bound protein was detected with rabbit polyclonal anti-CARDS toxin antibody and goat anti-rabbit HRP-conjugated polyclonal antibody, followed by development with TMB substrate. Wells with BSA alone served as negative controls, and the nonspecific bound values were subtracted from individual test scores. Values are means ± standard errors of the means (error bars) for triplicate wells from three separate experiments. No immunological cross-reactivity was observed between anti-CARDS toxin antibody and AnxA2.