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Transient OxyR oxidation by hydrogen peroxide in vitro. OxyR4C→A (1 μM final concentration) was reduced fully by incubation with a buffer containing 25 mM GSH, 0.1 mM GSSG, and 10 μM Grx1. Hydrogen peroxide (2 μM) was added, and the redox status of OxyR was assayed at 0.5, 10, 30, 60, 120, 180, and 240 min. Samples were mixed with a 1/10 vol of 100% TCA and then treated with AMS. Separation and detection of OxyR were achieved by SDS/PAGE and immunoblot analysis.
