Fig. 6. GSSG suppresses TNFα-stimulated NF-κB transactivation via induction of IKK-β glutathionylation.

a H₂O₂, BCNU, or diamide pretreatment suppressed NF-κB-mediated gene expression after TNFα stimulation. HepG2 cells were pretreated with H₂O₂, BCNU or diamide for 2 h before TNFα (40 ng/mL) addition. Total RNA was isolated, and NF-κB-targeted gene expression was determined by real-time RT-PCR. All values are expressed as the mean ± SD from three or more independent studies. *p < 0.05, **p < 0.01 versus TNFα-treated cells. b H₂O₂ pretreatment suppresses TNFα-induced nuclear p65/DNA-binding activity. HepG2 cells were pretreated with H₂O₂ (0.2 mM) for 2 h before TNFα (100 ng/mL) addition. Four hours later, nuclear fractions were isolated and subjected to ELISA for the measurement of p65/DNA-binding activities. All values are expressed as the mean ± SD from three or more independent studies. *p < 0.05 versus TNFα-treated cells. c H₂O₂ pretreatment abolished the TNFα-stimulated increase in IκB-α phosphorylation. HepG2 cells were pretreated with H₂O₂ (0.2 mM) for 2 h before TNFα (100 ng/mL) stimulation, and cell lysates were collected 15 min after TNFα exposure for western blot analysis of phosphorylated IκB-α. d H₂O₂ pretreatment inhibited TNFα-stimulated IKK-β activation/phosphorylation. HepG2 cells were pretreated with H₂O₂ for 2 h before TNFα (100 ng/mL) stimulation, and cell lysates were collected at the indicated time points for western blot analysis of the phosphorylated IKK-β levels. e H₂O₂, BCNU, or diamide pretreatment increased the IKK-β-SSG levels. HepG2 cells were treated with H₂O₂, BCNU, or diamide for 1 h, and cell lysates were collected and subjected to immunoprecipitation and western blot analysis for glutathionylated IKK- β. IB immunoblotting, IP immunoprecipitation