Fig. 7. Deactivation (reduction) of Yap1 by the thioredoxin system. (A and B) Expression of TRX2 (A) and TRR1 (B) in wild-type, Δtrr1 (A), Δtrx1Δtrx2 (B), Δgsh1Δglr1pro2-1 and Δtsa1 strains. Cells were treated with 300 µM H₂O₂ for the indicated time and processed for RT–PCR as in Figure 1A. The ACT1 autoradiograms are not shown. (C) Yap1 is oxidized in mutants of the thioredoxin system. yMyc-Yap1, Δtrx1Δtrx2 and Δtrr1 expressing Myc-Yap1 (grown to an OD₆₀₀ of 0.3) were processed as in Figure 2A and extracts were analysed by immunoblotting after non-reducing PAGE. (D) Reduction of oxidized Yap1 by the thioredoxin system. yMyc-Yap1 (grown to an OD₆₀₀ of 0.4), treated with 400 µM H₂O₂, lysed with glass beads in 100 mM Tris–HCl pH 8, 0.1% SDS, 1 mM EDTA, complete protease inhibitors and PMSF. Twenty-five micrograms of extracts were incubated with 1, 10 or 20 µM thioredoxin (from Spirulina sp.), 1.3 µM E.coli thioredoxin reductase and 1 mM NADPH (Sigma) for 30 min at 37°C. Iodoacetamide (75 mM) was added and the samples were analysed by immunoblotting after non-reducing PAGE.