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. 2008 Mar;19(3):1104–1112. doi: 10.1091/mbc.E07-10-1004

Figure 3.

Figure 3.

Celastrol activates the yeast oxidant-responsive transcription factor Yap1 through the carboxy-terminal domain. (A) Schematic of Yap1 cysteine residues involved in oxidation and alkylation. (B) Wild-type (BY4741) and gpx3Δ cells containing the Yap1 reporter (pCEP12) were treated with either no reagent, 300 μM H2O2, DMSO, or 10 μM celastrol. Harvested cells were subsequently assayed for β-galactosidase activity as described previously. (C) Cells expressing a TAP-tagged allele of YAP1 from the endogenous genomic locus were grown to logarithmic phase and treated with DMSO, 300 μM H2O2, or 10 μM celastrol for 10 min, followed by trichloroacetic acid precipitation. Cell extracts were prepared by glass-bead lysis in nonreducing sample buffer and either left untreated (−DTT) or reduced with 10 mM DTT (+DTT) before SDS-PAGE. Yap1 migration was detected using anti-protein A antibody. The locations of the Yap1-TAP fusion and the DTT-sensitive Yap1-TAP·Gpx3 heterodimer are indicated. (D) Cells expressing either wild-type or mutant GFP-Yap1 regulatory domain fusion proteins were treated with DMSO, 300 μM H2O2, or 10 μM celastrol to monitor GFP subcellular localization as described in Materials and Methods. Cartoon illustrates diffuse Yap1-GFP fluorescence before modification of the c-CRD (by either pathway), and nuclear localization after treatment. The yeast vacuole is indicated by a dark gray circle in the cartoon. Results are representative of three independent experiments.