FIG. 6.
Reduced expression of cytoprotective target genes and enhanced oxidative stress in skin and papillomas of dnNrf2 transgenic mice. (A) Samples of 20 μg total RNA from nontumorigenic skin (pools of five biopsy samples per genotype) and from papillomas (pools of five biopsy samples per genotype) of wild-type and transgenic mice were analyzed by RPA for the presence of NQO1, γ-GCSh, IL-6, IL-1β, Nrf2, and GAPDH mRNAs. Twenty micrograms of tRNA was used as a negative control. One thousand cpm of the hybridization probes was loaded in the lanes labeled “probe” and used as a size marker. Densitometric quantification of each RNase protection assay result (normalized to GAPDH) is shown on the right side. The strongest signal was arbitrarily set as 100. (B) Thirteen micrograms of total protein from untreated skin, from nontumorigenic skin which was treated for 20 weeks with DMBA/TPA, and from papillomas of wild-type and transgenic animals was analyzed for the presence of oxidized proteins by Oxyblot analysis. (C) Twenty micrograms of total protein from DMBA-treated skin of wild-type and transgenic mice and 7.5 μg total protein from skin, which was treated once with DMBA and once with TPA were analyzed for the presence of oxidized proteins by Oxyblot analysis. Staining of the same membrane with an antibody directed against β-actin served as a loading control in panel B. Staining with an antibody against lamin A served as a loading control in panel C.