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 together with the indicated concentrations of -factor for 90 min, and then -galactosidase activity was measured. Kainate Receptor Purity & Documentation Information are implies SEM from 3 experiments, each performed in quadruplicate. Information are expressed as a percentage of your -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; offered in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 4. Crosstalk in between mating and glucose-sensing pathways(A to C) Analysis in the effects of high 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 getting left untreated or treated with 3 -factor (-F) for the indicated occasions prior to they were harvested for evaluation. Top: Samples have been cIAP-2 Storage & Stability analyzed by Western blotting with antibody against phosphorylated p4442 MAPK (to detect p-Fus3 and p-Kss1), too as with antibodies particular for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was utilized as a loading handle. Middle: Densitometric analysis from the abundance of p-Fus3. Bottom: Densitometric evaluation with the abundance of total Fus3. For densitometric analysis, essentially the most intense band on each blot was set at one hundred , as well as the intensities of your other bands have been expressed as percentages in the maximum. Results 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 have been left untreatedSci Signal. Author manuscript; accessible 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 on the -galactosidase activity of pheromone-treated WT cells cultured in two glucose, which was set at 100 . Data are suggests SEM from 3 independent experiments, each and every 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 from the MAPKKK Ste11. Early og phase cells had been resuspended in medium containing either two or 0.05 glucose. Cells transformed with empty plasmid have been treated with three -factor for 5 min, whereas cells expressing STE11-4 were collected five min just 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 of your 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 two glucose was set at one hundred . Information are signifies SEM from 3 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. 5. Shmoo formation and mating are impaired below 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.
