Figure 6. Degradation of deposited C3b by B. anthracis spores-bound PLG.

(A) Spores (2×10⁷/well) were immobilized onto a microplate and incubated with 10% NHS (100 µl) for 30 min at room temperature. Washed spores were incubated with 2 µg/well PLG for 1 h in the presence or absence of protease cocktail. Bound PLG was activated by uPA (20 units/well) for 3 h at 37°C. Deposited C3b was detected by anti-C3c IgG and HRP-conjugated secondary antibody followed by TMB reaction. C3b deposition is expressed as the mean absorbance at 450 nm of quadruplicates. Error bars indicate ± standard deviation. *P<0.001 (paired Student's t-test). (B) Spores were incubated with plasmin and washed extensively as described in Figure 2A. C3b was incubated with the spores in the presence or the absence of leupeptin and C3b degradation was analyzed by Western blot with anti-C3c antibody.