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 were treated using the indicated concentrations of -factor for 90 min, and after that -galactosidase activity was measured. Data are suggests SEM from three experiments, each and every performed in quadruplicate. Data are expressed as a percentage in the -galactosidase activity of WT cells at the maximum concentration of pheromone. P 0.05.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; available in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. four. Crosstalk involving mating and glucose-sensing pathways(A to C) Evaluation with the effects of higher and low glucose around the abundance and phosphorylation of Fus3. (A) WT cells, (B) elm1sak1tos3 cells, and (C) reg1 cells had been cultured in medium containing two or 0.05 glucose for 5 min prior to becoming left untreated or treated with three -factor (-F) for the indicated instances ahead of they have been harvested for analysis. Prime: Samples had been analyzed by Western blotting with antibody against phosphorylated p4442 MAPK (to detect p-Fus3 and p-Kss1), as well as with antibodies distinct for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was applied as a loading control. Middle: Densitometric evaluation with the abundance of p-Fus3. Bottom: Densitometric analysis from the abundance of total Fus3. For densitometric evaluation, by far the most intense band on each and every blot was set at 100 , plus the intensities in the other bands were expressed as percentages of your maximum. Final results are suggests SEM from 3 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 of the -galactosidase activity of pheromone-treated WT cells cultured in 2 glucose, which was set at 100 . Information are indicates SEM from three independent experiments, each performed in quadruplicate. P 0.05. (E) WT cells had been transformed with empty plasmid or with plasmid encoding STE11-4, a constitutively ADAM10 Molecular Weight active mutant with the MAPKKK Ste11. Early og phase cells have been resuspended in medium containing either 2 or 0.05 glucose. Cells transformed with empty plasmid were treated with 3 -factor for five min, whereas cells expressing STE11-4 have been collected 5 min following resuspension in fresh medium. Samples have been analyzed by Western blotting with antibodies against phosphorylated p4442 MAPK and total Fus3. Bar graphs represent densitometric analysis in the intensities of bands corresponding to p-Fus3, Bak drug normalized to these corresponding to total Fus3. For every set of cells, the abundance of p-Fus3 in two glucose was set at 100 . Data are means SEM from three independent experiments.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; readily available in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. five. Shmoo formation and mating are impaired beneath circumstances of restricted glucose availability(A) Mating efficiency assay. Separate cultures of WT mating-type a cells (BY4741) and WT mating-type cells (BY4742) have been grown in medium containing 2 glucose. Cells (1 107) f.
