Figure 3. SDS-PAGE and Western blot analyses of secreted proteins produced by USA300 and isogenic variants after 8 h of growth in TSB, or TSB supplemented with 25 µM linoleic acid (A), and assay of total protease activity in culture supernatant (B).

For (A), protein loading was 2.0 OD₆₀₀ units for Coomassie staining, and 0.02 OD₆₀₀ units for Western blots, which were developed with primary antibody specific for Aur, and SspA as indicated. Arrows on the Coomassie stained gel indicate the selective induction of secreted proteases in response to linoleic acid. The arrow on the right margin indicates the position of proGeh. In (B), total protease activity in 8 h culture supernatant of USA300 and isogenic variants was determined with FITC-casein substrate. Cultures were grown with 25 µM linoleic acid as indicated, and assay buffer was supplemented with 10 mM EDTA where indicated, to inhibit metalloprotease. Data are reported as fluorescence emission at 535 nm (ε₅₃₅), measured in arbitrary fluorescence units.