Figure 4. IL-1β Is S-Glutathionylated In Vivo.
(A) Survival of mice treated with 6Gy whole-body γ-irradiation. The indicated amounts of recombinant mouse IL-1β were administered i.p. 20 hr before irradiation. Survival was analyzed using Kaplan-Meier survival curves and the log-rank test. *p < 0.005 versus control.
(B) H2O2 concentrations in the bone marrow (BM) of irradiated mice. Data are means ± SD of n = 3 mice. *p < 0.005 versus time 0.
(C) IL-1β-elicited ROS elevation in the BM. Data shown are means ± SD of n = 3 mice. *p < 0.005 versus time 0.
(D) S-glutathionylation of i.p.-injected recombinant mouse IL-1β detected by western blotting. His-tagged recombinant mouse IL-1β (1 μg/mouse) was i.p.-injected into WT mice. Whole-body γ-irradiation was conducted 24 hr after injection. Recombinant IL-1β was pulled down from the serum or BM lavage using Ni-NTA agarose beads at the times indicated after irradiation. Precipitated IL-1β was assessed by western blotting using anti-mouse IL-1β antibody. S-glutathionylation of recombinant IL-1β was detected using an anti-GSH antibody. Representative immunoblots are shown.
(E) Cysteine modification of i.p.-injected recombinant mouse IL-1β analyzed by MS. Recombinant IL-1β (1 μg) was injected 24 hr before irradiation and was pulled down from serum using Ni-NTA agarose beads 3 days after irradiation. Mice not injected with recombinant IL-1β were used as controls. Representative graphs of total intensity of precursor, precursor (M+1), and precursor (M+2) for each indicated modification are shown.