(A) The effect of glutathionylation on H2O2-induced irreversible oxidation of IL-1β assessed by MS. IL-1β was treated with or without GSSG (250 μM) and then H2O2 (100 μM) for another 30 min at 37°C. The cysteine modification was detected by MS, and the relative amount of each cysteine modification was assessed using Skyline software. Representative plots of precursor, precursor (M+1), and precursor (M+2) intensities for each indicated modification are shown. The molecular weights (MW) of peptides containing the indicated modified cysteine are shown.
(B) The effect of glutathionylation on H2O2-induced deactivation of IL-1β assessed by the IκB degradation assay. Indicated amounts of IL-1β were pretreated overnight with GSSG (250 μM) and then H2O2 (100 μM) for another 30 min at 37°C before adding to HeLa cell cultures. Representative immunoblots for IκBα are shown.
(C) Densitometry results. Shown are the means (±SD) of three independent experiments.