Transcription of UFO1 in response to arsenate, H2O2, and UV.

2012-02-23T01:53:10Z (GMT) by Anna Lavut Dina Raveh

A. Wild type cells were grown in SC 2% glucose medium overnight, diluted to A600 = 0.1, regrown to A600 = 0.5 and treated with 1 mM arsenate, 8.8 mM H2O2, or irradiated with 40 mJ/cm2 UV. Aliquots were collected at the indicated times and analyzed by qRT-PCR. mRNA levels were normalized to ACT1 and to time 0 (untreated log cells). B. pGAL-GFP-UFO1 was expressed in ufo1Δ mutants by overnight induction with 2% galactose. Next morning cells were diluted to A600 = 0.1, regrown to A600 = 0.5 then untreated, or stressed with 1 mM arsenate or 8.8 mM H2O2 for 30 minutes, or irradiated with 40 mJ/cm2 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 ACT1 and to time 0 (untreated log cells). C. Western blot analysis of Ufo1GFP protein produced from the tagged genomic UFO1-GFP gene. Anti-GFP antibodies were used to detect Ufo1GFP and anti-α-tubulin antibodies to detect α-tubulin that serves as a loading control. D. UFO1 mRNA levels in untreated wild type, yap1Δ or pdr1Δ mutants. mRNA levels were normalized to ACT1 and to w.t. mRNA levels. E. yap1Δ or pdr1Δ mutants grown, treated and analyzed as in Figure 1A. mRNA levels were normalized to ACT1 and to time 0 (untreated log cells).

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