results shown are representative of four (Panel A) and one (Panel B) experiments, respectively, of similar design. Bars indicate +/- SEM in triplicate. Statistical analysis was performed via one-way ANOVA using a Dunnett’s Multiple Comparison post-test (*** P < .001). Figure 3 εACA inhibits huPLG binding to FT in a dose-dependent fashion. FTLVS was coated onto microtiter plate wells and incubated for 2 hours with purified huPLG (3 μg/mL) in the presence or absence of titrated concentrations of εACA. The results shown are representative of 3 experiments of similar design. Bars indicate +/- SEM in triplicate. Statistical analysis performed via one-way ANOVA using a Kruskal-Wallis test determined a p-value of < 0.0001. Figure 4 PLG binds to the outer envelope of FT. Laser scanning confocal microscopy of PLG-associated RO4929097 chemical structure FTLVS was performed as described in “”Materials and Methods”". Bound huPLG ligand was detected using sheep anti-human PLG antibody followed by incubation with Dylight-488 conjugated donkey, anti-sheep/goat
IgG secondary antibody. Samples were visualized using a Zeiss LSM 510 confocal microscope. Plasmin activation on the surface of FT LVS in vitro by a PLG activator In other bacterial systems, surface-bound PLG can be converted to its proteolytically active plasmin form that contributes to the organism’s virulence [21–24]. To test whether huPLG bound to FTLVS can be converted to plasmin, we used a chromogenic plasmin substrate (H-D-Val-Leu-Lys-pNA) to detect proteolytic activity following the addition of tissue Selleckchem C188-9 PLG activator (tPA) (Figure 5). We also found that plasmin on the surface of FT can break down fibronectin (Figure 6), suggesting that FT-bound plasmin can potentially participate in the degradation of extracellular matrices. Figure 5 FT surface-bound huPLG can be
converted to plasmin. Adenosine FTLVS was incubated with huPLG at a concentration of 96 μg/mL. After removal of unbound huPLG, a chromogenic plasmin substrate (D-VLK-pNA), tissue PLG activator (tPA), or both were then added to test the proteolytic ability of each sample preparation. Semaxanib manufacturer Conversion of the chromogenic substrate was measured by comparison of Δ405 nm. The results shown are representative of 3 experiments of similar design. Bars indicate +/- SEM in triplicate. Statistical analysis was performed via one-way ANOVA using a Dunnett’s Multiple Comparison post-test (*** P < .001). Figure 6 Fibronectin is a substrate for plasmin bound to FT. FTLVS (109 CFU) were incubated with 100 μg of huPLG and 0.5 μg tissue tPA for 1 hour at 37°C. After removal of unbound huPLG and tPA, 3 μg fibronectin was added and allowed to incubate for 24 hours at 37°C. Supernatant from each preparation were separated by SDS-PAGE and transferred to PVDF membrane. Degradation of fibronectin was detected by Western blot analysis as described in “”Materials and Methods”".