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 had been treated with all the indicated concentrations of -factor for 90 min, then -galactosidase activity was measured. Information are implies SEM from three experiments, each performed in quadruplicate. Information are IL-35 Protein site expressed as a percentage of the -galactosidase activity of WT cells at the IL-10 Protein Gene ID maximum concentration of pheromone. P 0.05.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; offered in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 4. Crosstalk among mating and glucose-sensing pathways(A to C) Analysis from 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 2 or 0.05 glucose for five min just before being left untreated or treated with three -factor (-F) for the indicated times before they were harvested for analysis. Best: 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 specific for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was utilized as a loading manage. Middle: Densitometric analysis of your abundance of p-Fus3. Bottom: Densitometric analysis on the abundance of total Fus3. For densitometric evaluation, probably the most intense band on each blot was set at 100 , along with the intensities with the other bands have been expressed as percentages with the maximum. Benefits are signifies 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; obtainable 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 one hundred . Data are implies SEM from three independent experiments, each performed in quadruplicate. P 0.05. (E) WT cells have been transformed with empty plasmid or with plasmid encoding STE11-4, a constitutively active mutant with the MAPKKK Ste11. Early og phase cells were resuspended in medium containing either two 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 five min soon after resuspension in fresh medium. Samples had been analyzed by Western blotting with antibodies against phosphorylated p4442 MAPK and total Fus3. Bar graphs represent densitometric evaluation from the intensities of bands corresponding to p-Fus3, normalized to those corresponding to total Fus3. For every single set of cells, the abundance of p-Fus3 in 2 glucose was set at one hundred . Information are signifies SEM from three independent experiments.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; offered 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 conditions 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.
