Ysis of pheromone-dependent gene transcription in WT and reg1 cells. Cells
Ysis of pheromone-dependent gene transcription in WT and reg1 cells. Cells expressing a FUS1-lacZ reporter have been treated together with the indicated concentrations of -factor for 90 min, and then -galactosidase activity was measured. Data are suggests SEM from three experiments, each and every performed in quadruplicate. Information are expressed as a percentage from the -galactosidase activity of WT cells in the maximum concentration of pheromone. P 0.05.NIH-PA Author JNK1 Accession Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; out there in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 4. Crosstalk involving mating and glucose-sensing pathways(A to C) Caspase 4 Source evaluation from the effects of high and low glucose around the abundance and phosphorylation of Fus3. (A) WT cells, (B) elm1sak1tos3 cells, and (C) reg1 cells were cultured in medium containing two or 0.05 glucose for five min before becoming left untreated or treated with three -factor (-F) for the indicated times just before they were harvested for evaluation. Major: Samples had been analyzed by Western blotting with antibody against phosphorylated p4442 MAPK (to detect p-Fus3 and p-Kss1), at the same time as with antibodies specific for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was made use of as a loading control. Middle: Densitometric analysis with the abundance of p-Fus3. Bottom: Densitometric analysis on the abundance of total Fus3. For densitometric analysis, probably the most intense band on each and every blot was set at one hundred , and also the intensities on the other bands had been expressed as percentages on the maximum. Results are suggests SEM from three independent experiments. (D) Evaluation of pheromone-dependent gene transcription in WT, elm1sak1tos3, and reg1 cells expressing a FUS1-lacZ reporter that were left untreatedSci Signal. Author manuscript; offered in PMC 2014 July 23.NIH-PA Author ManuscriptClement et al.Pageor treated with 30 -factor for 90 min in medium containing either two or 0.05 glucose. Information are expressed as percentages on the -galactosidase activity of pheromone-treated WT cells cultured in 2 glucose, which was set at 100 . Data are indicates SEM from three independent experiments, every single performed in quadruplicate. P 0.05. (E) WT cells were transformed with empty plasmid or with plasmid encoding STE11-4, a constitutively active mutant from the MAPKKK Ste11. Early og phase cells have been resuspended in medium containing either 2 or 0.05 glucose. Cells transformed with empty plasmid have been treated with 3 -factor for 5 min, whereas cells expressing STE11-4 had been collected 5 min immediately after resuspension in fresh medium. Samples were analyzed by Western blotting with antibodies against phosphorylated p4442 MAPK and total Fus3. Bar graphs represent densitometric evaluation on the intensities of bands corresponding to p-Fus3, normalized to those corresponding to total Fus3. For every set of cells, the abundance of p-Fus3 in two glucose was set at 100 . Data are implies SEM from 3 independent experiments.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; accessible in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 5. Shmoo formation and mating are impaired beneath situations of limited glucose availability(A) Mating efficiency assay. Separate cultures of WT mating-type a cells (BY4741) and WT mating-type cells (BY4742) had been grown in medium containing two glucose. Cells (1 107) f.
