FIG 5.

Determination of the overall protein synthesis via puromycin integration and growth of H₂O₂-treated S. oligofermentans. (A) Mid-exponential-phase cells of anaerobically grown S. oligofermentans were treated with 0.5 mM H₂O₂ for indicated times (left panel) or with various concentrations of H₂O₂ for 30 min (right panel). Puromycin was added at a final concentration of 10 μg/ml 10 min before cell collection, and then cells were lysed. Same amounts of total cell protein were loaded on two 12% SDS-PAGE gels, and one was examined by Western blotting using anti-puromycin antibody (upper panel) and the other was stained with Coomassie brilliant blue as an indicator of equivalent loading (lower panel). Molecular weight marker is shown on the left. (B) S. oligofermentans was grown anaerobically until the OD₆₀₀ reached 0.4, and cells were divided into four aliquots, with three added with 40, 80 and 500 μM H₂O₂, respectively, and the other left untreated. The OD₆₀₀ was measured at indicated time points. Arrow indicates the time point of H₂O₂ addition. Triplicate experiments were performed, and either one representative result is shown (A) or the averages ± SD are shown (B).