Figure 7.

Overexpression of Nrf2-dependent HO-1 inhibits NF-κB activation and translocation. A: Cells were stimulated with TNF-α for the indicated time intervals. The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser⁵³⁶) or anti-phospho-IκBα (Ser³²) Ab. The cytosolic and nuclear extracts were prepared and subjected to Western blot using an anti-p65 or anti-IκBα Ab (left). Cells were pretreated with helenalin (1 μmol/L), PP1 (10 μmol/L), NAC (10 mmol/L), DPI (10 μmol/L), or APO (100 μmol/L), and then treated with TNF-α for 1 hour. The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser⁵³⁶) Ab (right). B, C: Cells were pretreated with CoPP IX for 16 hours in the presence or absence of ZnPP IX, and then treated with TNF-α for 1 hour. B: The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser⁵³⁶) or anti-phospho-IκBα (Ser³²) Ab. The cytosolic and nuclear extracts were prepared and subjected to Western blot using an anti-p65 or anti-IκBα Ab. C: Cells were fixed, and then labeled with anti-p65 Ab and fluorescein isothiocyanate-conjugated secondary Ab. Individual cells were imaged as described in the Materials and Methods. D: Cells were transiently transfected with a NF-κB-luc reporter gene, and then treated with TNF-α for the indicated time intervals (left) or for 1 hour in the presence or absence of CoPP IX or ZnPP IX (right). The luciferase activity of NF-κB was measured. Data represent the mean ± SEM from at least three independent experiments. *P < 0.05; ^#P < 0.01 as compared with the basal level (left). Significant differences between the compared groups are indicated: *P < 0.05; ^#P < 0.01 (right).