FIG. 3.

Incubation conditions affecting the stability of rExoU and phospholipase activity. (A) The degradation of rExoU (1 μg) during incubation at pH 7.4 or 6.5 at 30°C in the presence (U ext) or absence (U) of yeast extract (5 μg) was monitored by Western blotting. Aprotinin (2.3 μg) was added to the incubation mixture at pH 6.5 to minimize rExoU degradation. The samples were used after 1 or 3 h of incubation for the in vitro assay. For positive controls, 1 μg of rExoU or 5 μg of extract was incubated individually, and fresh extract or rExoU was added immediately prior to the assay. Percentages indicate levels of rExoU activity relative to those exhibited by positive controls. (B) The incubation temperature of in vitro assays was varied, and rExoU activity was quantified in the presence of a protease inhibitor. Samples containing 5 μg of rExoU, 5 μg of yeast extract proteins, and 6 μg of aprotinin were incubated with liposomes for 15 min.