The abundance of Srx protein was not affected by exposure of any of these cells to 100 mM ethanol for 18 hours, whereas the protein levels of Srx were increased slightly in E47 cells (Supporting Information Fig. 2B). Similar treatment of primary mouse hepatocytes also showed no significant effect of ethanol on Srx and CYP2E1 expression (Supporting Information Fig. 2C). It was shown previously that HepG2 cells resist the adverse effect of

ethanol because the cells contain a very low amount of CYP2E1.39 Chronic ethanol feeding of mice was previously shown to increase Nrf2 expression ≈2-fold in the liver.6 The role of Nrf2 in ethanol-induced Autophagy inhibitor Srx expression in the liver was investigated with the use of Nrf2-deficient mice. The amount of Srx protein in the liver was increased ≈9-fold by ethanol feeding in Nrf2+/+ mice but only ≈2-fold in Nrf2−/− mice (Fig. 2B,C). Ethanol feeding also induced similar changes in the hepatic abundance of Srx mRNA (Fig. 2D). In addition, the basal level of Srx mRNA was reduced by ≈50% in Nrf2−/− mice compared with that in Nrf2+/+ animals (Fig. 2D). These results suggested that the Nrf2-ARE pathway plays a key role in the induction of Srx in the liver of ethanol-fed mice. The observation that ethanol still induced an ≈2-fold increase in Srx expression in the liver of Nrf2−/− mice, however, suggested that the AP-1-ARE pathway might also Fostamatinib contribute to this effect. Srx is responsible for

reduction of the Florfenicol hyperoxidized forms of 2-Cys Prx enzymes (Prx I to IV) generated during elimination of peroxides. Hyperoxidized 2-Cys Prxs can be detected by immunoblot analysis with antibodies generated in response to a sulfonylated peptide modeled on the conserved peroxidatic cysteine residue (CP). Given that the amino acid sequences surrounding CP are identical for 2-Cys Prx enzymes, the antibodies react with all of these hyperoxidized proteins.13 To investigate the role of Srx in ethanol-fed mice, we generated Srx−/− mice (Supporting Information Fig. 3). Srx+/+ and

Srx−/− mice were then subjected to chronic ethanol feeding, after which liver proteins were subjected to immunoblot analysis with antibodies to Srx, to sulfinic forms of 2-Cys Prxs (Prx-SO2), and to Prx I to IV (Fig. 3). Prx I and Prx II, which differ by only one amino acid residue in size, cannot be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the molecular sizes of Prx I/II, III, and IV differ sufficiently to allow such separation. In NIH 3T3 cells that had been exposed to 100 μM H2O2 for 10 minutes the cytosolic enzymes Prx I and II as well as the mitochondrial enzyme Prx III were found to be completely hyperoxidized (see below), whereas hyperoxidation of the ER-localized Prx IV was not detected (not shown). Extracts of the H2O2-treated cells are included as a standard for Prx I/II-SO2 and Prx III-SO2 in Figure 3.