The biotinylated rFbp were dissolved in BVBS containing 0.02% (v/v) Tween 20. The binding of biotinylated Fn to III1-C in the presence of 1 μg or 10 μg rFbp was measured. Absorbance at 280 nm was used to calculate the protein concentration of Fn and

Fn fragments using ɛpercent= 10. The concentration of rFbp was measured by the Bradford method (Bio-Rad, Hercules, CA, USA) using BSA as a standard. All experiments were performed in triplicate. Statistical significance (P < 0.05, P < 0.01) was determined by comparison with controls using Student's two-tailed t-test. To determine which Fn fragments are recognized by the rFbp, a plate binding assay was performed in which binding of biotinylated-rFbpA or -rFbpB to immobilized Fn fragments (70 kDa, 30 kDa, 45 kDa, 110 kDa or selleck chemicals III1-C) was assayed. The Fn fragments were mapped according to their position within the Fn polypeptide (Fig. 1a). Of the Fn fragments tested, both rFbpA and rFbpB bound only to the III1-C fragment of Fn (Fig. 1b). Both rFbpA and rFbpB were found to bind to the III1-C fragment. However, the III1-C fragment of serum Fn is known to be cryptic. Therefore, rFbp-binding proteins from Fn were purified by affinity chromatography on rFbpA- and rFbpB-Sepharose columns. Following

elution of bound proteins with 4 M urea, the yield of affinity purified binding protein from rFbpA-Sepharose and rFbpB-Sepharose chromatography was 0.96% and 1.08% of the applied Fn protein, respectively. In order to characterize the purified rFbp-BP, epitope mapping with various anti-Fn mAbs using immobilized Apitolisib research buy Fn fragments in a plate binding assay was first carried out.

for The mAb HB91 reacted strongly with both the N-terminal 70-kDa and 30-kDa fragments of Fn, but reacted weakly with the 45-kDa fragment. The other three mAbs tested, HB39, ZET1, and ZET2, reacted with the 110-kDa Fn fragment. The HB39 mAb was the only mAb that also reacted weakly with both the N-terminal 70-kDa and 30-kDa fragments (Fig. 2a). No mAb tested here reacted with III1-C (Fig. 2b). To determine if the rFbp-BP might contain Fn-epitopes, whether the rFbp-BP were recognized by the anti-Fn mAbs, using SDS-PAGE and Western blotting analysis was checked. Silver staining of SDS-gels showed that both rFbpA-BP and rFbpB-BP consisted of a major, slightly broad protein band with a size of 450 kDa, and minor bands with the sizes of 180, 160 and 84 kDa (Fig. 3a and b). When binding of the anti-Fn mAbs was tested by Western blotting, the 450-kDa protein band of the rFbp-BP reacted with both HB91 and HB39, but not with ZET1 or ZET2. To determine whether rFbp-BP expressed III1-C, a rFbp-binding assay to rFbp-BP in the presence of III1-C peptides was performed. Binding of both rFbpA and rFbpB to rFbpA-BP and rFbpB-BP, respectively, was significantly inhibited by the presence of III1-C peptide in a dose-dependent manner (Fig. 4).