Transcription of <i>UFO1</i> in response to arsenate, H<sub>2</sub>O<sub>2</sub>, and UV.

2012-02-23T01:53:10Z (GMT) by Anna Lavut Dina Raveh
<p>A. Wild type cells were grown in SC 2% glucose medium overnight, diluted to <i>A</i><sub>600</sub> = 0.1, regrown to <i>A</i><sub>600</sub> = 0.5 and treated with 1 mM arsenate, 8.8 mM H<sub>2</sub>O<sub>2</sub>, or irradiated with 40 mJ/cm<sup>2</sup> UV. Aliquots were collected at the indicated times and analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells). B. p<i>GAL-GFP-UFO1</i> was expressed in <i>ufo1Δ</i> mutants by overnight induction with 2% galactose. Next morning cells were diluted to <i>A</i><sub>600</sub> = 0.1, regrown to <i>A</i><sub>600</sub> = 0.5 then untreated, or stressed with 1 mM arsenate or 8.8 mM H<sub>2</sub>O<sub>2</sub> for 30 minutes, or irradiated with 40 mJ/cm<sup>2</sup> UV. The cells were washed and transferred to SC with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells). C. Western blot analysis of Ufo1<sup>GFP</sup> protein produced from the tagged genomic <i>UFO1-GFP</i> gene. Anti-GFP antibodies were used to detect Ufo1<sup>GFP</sup> and anti-α-tubulin antibodies to detect α-tubulin that serves as a loading control. D. <i>UFO1</i> mRNA levels in untreated wild type, <i>yap1Δ</i> or <i>pdr1Δ</i> mutants. mRNA levels were normalized to <i>ACT1</i> and to w.t. mRNA levels. E. <i>yap1Δ</i> or <i>pdr1Δ</i> mutants grown, treated and analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002527#pgen-1002527-g001" target="_blank">Figure 1A</a>. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells).</p>

Categories

License

CC BY 4.0