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, then -galactosidase activity was measured. Information are indicates SEM from 3 experiments, every performed in quadruplicate. Data are expressed as a percentage with 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; accessible 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) 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 have been cultured in medium containing 2 or 0.05 glucose for 5 min prior to becoming left untreated or treated with three -factor (-F) for the indicated occasions ahead of they had been harvested for evaluation. Leading: Samples have 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 precise for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was utilized as a loading control. Middle: CB1 drug Densitometric evaluation of your abundance of p-Fus3. Bottom: Densitometric analysis from the abundance of total Fus3. For densitometric analysis, essentially the most intense band on every single blot was set at 100 , as well as the intensities from the other bands had been expressed as percentages of your maximum. Benefits are indicates SEM from three independent experiments. (D) Analysis of pheromone-dependent gene transcription in WT, elm1sak1tos3, and reg1 cells expressing a FUS1-lacZ reporter that had been 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. Data are expressed as percentages of the -galactosidase activity of BRDT Compound pheromone-treated WT cells cultured in 2 glucose, which was set at 100 . Data are indicates SEM from 3 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 on the MAPKKK Ste11. Early og phase cells have been resuspended in medium containing either two or 0.05 glucose. Cells transformed with empty plasmid were treated with 3 -factor for 5 min, whereas cells expressing STE11-4 were collected five 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 of the intensities of bands corresponding to p-Fus3, normalized to those corresponding to total Fus3. For each and every set of cells, the abundance of p-Fus3 in 2 glucose was set at 100 . Data are indicates SEM from three 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. 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) have been grown in medium containing 2 glucose. Cells (1 107) f.
