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 together with the indicated concentrations of –factor for 90 min, after which -galactosidase activity was measured. Information are means SEM from 3 experiments, each and every performed in quadruplicate. Information are expressed as a percentage of the -galactosidase activity of WT cells in the maximum concentration of pheromone. P 0.05.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 ManuscriptFig. four. Crosstalk in between mating and glucose-sensing pathways(A to C) 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 had been cultured in medium containing two or 0.05 glucose for 5 min just before being left untreated or treated with three -factor (-F) for the indicated instances just before they have been harvested for evaluation. Best: Samples have been analyzed by Western blotting with antibody against phosphorylated p4442 MAPK (to detect p-Fus3 and p-Kss1), also as with ALDH1 Formulation antibodies specific for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was used as a loading control. Middle: Densitometric evaluation of your abundance of p-Fus3. Bottom: Densitometric analysis of the abundance of total Fus3. For densitometric analysis, one of the most intense band on each and every blot was set at 100 , and the intensities with the other bands were expressed as percentages from the maximum. Outcomes are ALK1 Compound indicates SEM from 3 independent experiments. (D) Analysis of pheromone-dependent gene transcription in WT, elm1sak1tos3, and reg1 cells expressing a FUS1-lacZ reporter that were left untreatedSci Signal. Author manuscript; available in PMC 2014 July 23.NIH-PA Author ManuscriptClement et al.Pageor treated with 30 -factor for 90 min in medium containing either 2 or 0.05 glucose. Information are expressed as percentages with the -galactosidase activity of pheromone-treated WT cells cultured in two glucose, which was set at 100 . Information are signifies SEM from 3 independent experiments, each performed in quadruplicate. P 0.05. (E) WT cells were transformed with empty plasmid or with plasmid encoding STE11-4, a constitutively active mutant of your MAPKKK Ste11. Early og phase cells had been resuspended in medium containing either two or 0.05 glucose. Cells transformed with empty plasmid had been treated with 3 -factor for five min, whereas cells expressing STE11-4 have been collected 5 min just after 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 from the intensities of bands corresponding to p-Fus3, normalized to these corresponding to total Fus3. For each set of cells, the abundance of p-Fus3 in two glucose was set at one hundred . Data are indicates SEM from 3 independent experiments.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 Manuscript NIH-PA Author ManuscriptFig. 5. Shmoo formation and mating are impaired under circumstances of restricted 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.
