Fig. 4.

Mild oxidative stress directly regulates Nrf2 transcriptional activity through the Neh5 domain. A) Astrocytes within mixed astrocyte/neuronal cultures were co-transfected with Gal4-luc plasmid and either of the Nrf2-GBD fusion plasmids, plus pTK-renilla. Treatment with H₂O₂ (50 μM) for 8h was carried out 6 days post transfection (DIV08) before assessing luciferase reporter activity. Luciferase activity levels were normalized to Renilla and presented relative to GBD-Nrf2 control. *P < 0.05, significantly different from GBD-Nrf2 control, two-way ANOVA with Tukey's post-hoc test, (n = 3–10). B) Experiment performed as in (A) employing GBD-Neh5 (WT) and C191A mutant. *P < 0.05, significantly different from GBD-Neh5 control, two-way ANOVA with Tukey's post-hoc test, main effect of mutation was detected (GBD-Neh5-control Vs GDB-Neh5(C191A)) (two-way ANOVA, *P < 0.05), (n = 3–9). C) Astrocytes within mixed astrocyte/neuronal cultures were co-transfected with an ARE-dependent luciferase reporter, plus either WT or mutant Nrf2, or β-globin control, then stimulated ± H₂O₂ for 8h Luciferase activity levels were normalized to Renilla and presented relative to the unstimulated β-globin control (which relies on only endogenous Nrf2 to drive the reporter). *P < 0.05 two-way ANOVA, with Sidak's post-hoc test (n = 4).