Figure 6. Localization of SpsD binding sites in Fn.

(A), native Fn, the N29, the GBD, the recombinant fragments Fn_607–1263, Fn_1266–1908 and the C-terminal region Fn_1913–2477 of Fn were immobilized onto a microtiter plate and then incubated with of SpsD_167–519. Bound protein was detected by addition to the wells of anti-SpsD_37–519 mouse antibody, followed by HRP-conjugated rabbit anti-mouse antibody. Binding of FnBPB_163–480 to Fn and its fragments reported as control was detected by addition to the wells of anti-FnBPB_163–480 rabbit IgG, followed by HRP-conjugated goat anti-rabbit antibody. (B), different amounts of Fn and Fn fragments (1000-62.5 ng) were dotted onto a nitrocellulose membrane, probed with SpsD_167–519 (1 µg/ml) and complexes detected by addition of mouse anti-SpsD_37–519 IgG, followed by HRP-conjugated rabbit anti-mouse IgG. (C), surface-coated Fn, the N29 fragment and the region including the aa residues 1266–1908 were incubated with increasing concentrations of SpsD_167–519 and complex formation detected as reported in (A). Results shown in panels A and C are the mean values of triplicate samples ± S.E. The experiments were repeated three times with similar results.