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 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. Information 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; readily available in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. four. Crosstalk amongst mating and glucose-sensing pathways(A to C) Evaluation of your effects of high 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 five min ahead of getting left untreated or treated with 3 -factor (-F) for the indicated instances prior to they were harvested for evaluation. Top rated: Samples were analyzed by Western blotting with antibody against phosphorylated p4442 MAPK (to detect p-Fus3 and p-Kss1), at the same time as with antibodies distinct 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 evaluation on the abundance of total Fus3. For densitometric analysis, probably the most intense band on each and every blot was set at 100 , as well as the intensities of the other bands had been expressed as percentages of your maximum. CCR2 Storage & Stability 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; 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. Data are expressed as percentages in the -galactosidase activity of pheromone-treated WT cells cultured in 2 glucose, which was set at 100 . Information are suggests SEM from 3 independent experiments, every performed in quadruplicate. P 0.05. (E) WT cells had been transformed with empty Kainate Receptor drug plasmid or with plasmid encoding STE11-4, a constitutively active mutant on the MAPKKK Ste11. Early og phase cells were resuspended in medium containing either two or 0.05 glucose. Cells transformed with empty plasmid were treated with three -factor for 5 min, whereas cells expressing STE11-4 were collected five min immediately 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 analysis of the intensities of bands corresponding to p-Fus3, normalized to those corresponding to total Fus3. For each set of cells, the abundance of p-Fus3 in 2 glucose was set at one hundred . Information are suggests 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. five. Shmoo formation and mating are impaired under conditions of limited glucose availability(A) Mating efficiency assay. Separate cultures of WT mating-type a cells (BY4741) and WT mating-type cells (BY4742) were grown in medium containing two glucose. Cells (1 107) f.
