Fig. 2. In vivo oxidation of Yap1. (A) yMyc-Yap1 cultures (grown to an OD₆₀₀ of 0.4) before and after 2.5 min treatment with 400 µM H₂O₂ (lanes 1 and 2) or 1 mM t-butyl hydroperoxide (lanes 5 and 6) were lysed using TCA, treated with iodoacetamide and immunoblotted after non-reducing SDS–PAGE. Lanes 3 and 4 are the same as lanes 1 and 2 except that the TCA-precipitated protein pellet was dissolved in the presence of 200 mM DTT, before adding a 3.2 M excess of iodoacetamide. (B) Time course of Yap1 oxidation by H₂O₂. A yMyc-Yap1 culture before and 2.5, 5, 15, 30, 45 and 60 min after treatment with 400 µM H₂O₂, processed as in (A) (lanes 1 and 2), except that Yap1 was dephosphorylated and separated under non-reducing or reducing conditions as indicated. (C) The minimum H₂O₂ concentration required to oxidize Yap1 in vivo. A yMyc-Yap1 culture treated for 5 min with 25, 50, 100, 150, 200, 300 and 800 µM H₂O₂ and processed as in (A) (lanes 1 and 2). (D) Time-course analysis of Yap1 redox status in response to diamide. A yMyc-Yap1 culture before and 5, 15, 30, 45 and 60 min after treatment with 2 mM diamide, processed as in (A) (lanes 1 and 2) and separated under non-reducing or reducing conditions as indicated.