FIGURE 5.

Overexpression of Nrf2 up-regulates endogenous and transfected Cul3 and Rbx1 gene expression. A, Western analysis of Flp-in T-REx 293 (293) cells or 293/FRT/FLAG-Nrf2 (FRT/FLAG-Nrf2) cells expressing tetracycline-induced FLAG-tagged Nrf2 were incubated with 4 μg/ml tetracycline (TET) for the indicated times. Cells were harvested, lysed, and probed with anti-FLAG, anti-Cul3, and anti-Rbx1. Anti-β-actin was used as loading control. Densitometry measurements of bands were quantitated and shown in graph blots below. B and C, Cul3 and Rbx1 gene expression was analyzed by real time-PCR. 293 cells or FRT/FLAG-Nrf2 cells were seeded in a monolayer and treated with 4 μg/ml tetracycline for indicated time points. RNA was extracted, converted to cDNA. 50 ng of cDNA was analyzed using primers and probes specific for Cul3 and Rbx1 mRNA. Tetracycline-induced Nrf2 expression was also confirmed using specific primers for exogenous Nrf2. NQO1 and INrf2 were used as positive control, respectively. GusB primers and probes were used as internal control. D, 293 cells or FRT/FLAG-Nrf2 cells were co-transfected with Cul3 or Rbx1 ARE plasmids and the internal control plasmid pRL-TK. Twenty-four hours after transfection, the cells were treated with 4 μg/ml tetracycline for 8 or 16 h. pGL2 vector was used as negative control. The cells were harvested and analyzed for luciferase activity. The data shown are mean ± S.D. of three independent transfection experiments.