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. 2016 Mar 29;7(2):e00331-16. doi: 10.1128/mBio.00331-16

FIG 1 .

FIG 1 

The lack of antioxidant gene expression following combinatorial stress is accompanied by a differentially oxidized form of the Cap1 transcription factor. (A) Cap1 does not accumulate in the nucleus following combinatorial stress. Localization of Cap1 was detected by fluorescence microscopy of cells expressing Cap1-GFP (JC1060) under non-stress conditions (ns) and after exposure to 5 mM H2O2 or 5 mM H2O2 plus 1 M NaCl for 10 min. The position of the nuclei is shown by DAPI staining. (B) Cap1 is phosphorylated following H2O2 exposure but not combinatorial stress. Lysates from cells expressing 2Myc- and 6His-tagged Cap1 (Cap1-MH [JC948]), before (ns) and after the indicated stress treatments, were analyzed by Western blotting using an anti-Myc antibody. The positions of nonphosphorylated (Cap1) and phosphorylated (Cap1P) Cap1 are indicated. (C) Combinatorial stress inhibits H2O2-induced antioxidant gene expression. Northern blot analysis of RNA isolated from wild-type (Wt [JC747]) cells before (ns) and following a 10-min treatment with 5 mM H2O2, 1 M NaCl, or 5 mM H2O2 plus 1 M NaCl. Blots were analyzed with probes specific for the catalase (CAT1) and thioredoxin reductase (TRR1) genes. A probe against ACT1 was used as the loading control. (D) Diagram illustrating the detection of protein oxidation by AMS binding to reduced thiols. (E) Cap1 is differentially oxidized following combinatorial stress. Cap1 oxidation was analyzed by nonreducing SDS-PAGE and Western blotting of AMS-modified or untreated proteins prepared from cells expressing Cap1-HM before (ns) and following the stress treatments described above. Extracts from cap1Δ cells were included as a control. The positions of reduced (Cap1RED), oxidized (Cap1OX), and differentially oxidized (Cap1OX-1) Cap1 are indicated. (F) Differential oxidation of Cap1 is dependent on Gpx3. Cap1 oxidation was determined as described above in wild-type (Wt [JC948]) and gpx3Δ (JC1311) cells expressing Cap1-MH before (ns) and following the stress treatments described above. (G) Differential oxidation of Cap1 is dependent on Ypb1. Cap1 oxidation was determined as described above in wild-type (Wt [JC948]) and ybp1Δ (JC954) cells.