FIGURE 5.

H₂O₂ induces Nrf2-dependent proteasome activity in NCM460 cells. A, NCM460 cells were transiently treated with H₂O₂ at 200 μm, and nuclear extracts were analyzed by immunoblotting using Nrf2 and Hsp90 antibodies (left panel, a representative blot; middle panel, normalized Nrf2 band intensities from three independent experiments; means ± S.D.), or NCM460 cells were transfected with pARE or the control (pGL3) firefly luciferase reporter vector together with ptkRL prior to H₂O₂ treatment, and then cells were submitted to dual luciferase assay (right panel, data from three independent experiments performed in duplicate are shown). B, NCM460 cells were transiently treated with H₂O₂ at 200 μm, and reverse transcribed mRNA was submitted to real time PCR using S5a, α5, and β-actin primers (upper panel, mean values from three independent experiments performed in duplicate), or cellular extracts were analyzed by immunoblotting using S5a, α5, and Hsp90 antibodies (lower panel, a representative blot and normalized band intensities from three representative experiments; means ± S.D.). C, untreated NCM460 cells (left panel) or NCM460 cells pretreated with control, Nrf1, and Nrf2 siRNA (right panel) were transiently exposed to H₂O₂ at 200 μm and then submitted to the Suc-LLVY-AMC assay; data (means ± S.D.) from three independent experiments are shown. D, NCM460 cells treated with control, Nrf1, and Nrf2 siRNA and then subjected to treatment with 200 μm H₂O₂ for 24 h were analyzed by S5a and α5 Western blotting. A representative blot (left panel) and the normalized S5a and α5 band intensities (right panel) from three independent experiments as determined by band densitometry are shown (means ± S.D.). E and F, NCM460 cells were analyzed by ARE luciferase assay and Suc-LLVY-AMC assay after treatment with the indicated stimuli for 24 h; data (means ± S.D.) from three (E) and five (F) independent experiments performed in duplicate are shown. *, p < 0.05 compared with untreated.