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 AMPA Receptor manufacturer reporter have been treated with the indicated concentrations of –factor for 90 min, and then -galactosidase activity was measured. Data are signifies SEM from 3 experiments, 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. 4. Crosstalk among mating and glucose-sensing pathways(A to C) Evaluation on the effects of higher and low glucose on the abundance and phosphorylation of Fus3. (A) WT cells, (B) elm1sak1tos3 cells, and (C) reg1 cells were cultured in medium containing 2 or 0.05 glucose for five min before being left untreated or treated with 3 -factor (-F) for the indicated occasions just before they had been harvested for evaluation. Leading: 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 certain for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was employed as a loading handle. Middle: Densitometric analysis on the abundance of p-Fus3. Bottom: Densitometric evaluation of your abundance of total Fus3. For densitometric analysis, one of the most intense band on every blot was set at 100 , along with the intensities of the other bands were expressed as percentages on the maximum. Results are implies SEM from three independent experiments. (D) Analysis of pheromone-dependent gene transcription in WT, elm1sak1tos3, and reg1 cells expressing a FUS1-lacZ reporter that have been 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 two or 0.05 glucose. Data are expressed as percentages of your -galactosidase activity of pheromone-treated WT cells cultured in two glucose, which was set at 100 . Information are means 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 in the MAPKKK Ste11. Early og phase cells have been resuspended in medium containing either 2 or 0.05 glucose. Cells transformed with empty plasmid have been treated with 3 -factor for five min, CCKBR custom synthesis whereas cells expressing STE11-4 had been collected five min 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 two 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; obtainable 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 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.
