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. Information are signifies SEM from three experiments, each performed in quadruplicate. Information are expressed as a percentage of the -galactosidase activity of WT cells at 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 amongst mating and glucose-sensing pathways(A to C) Analysis in 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 two or 0.05 glucose for five min ahead of being left untreated or treated with three -factor (-F) for the indicated times prior to they were harvested for analysis. Best: Samples have been Cyclophilin A, Mouse (tag free) analyzed by Western blotting with antibody against phosphorylated p4442 MAPK (to detect p-Fus3 and p-Kss1), as well as with antibodies particular for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was employed as a loading manage. Middle: Densitometric analysis of the abundance of p-Fus3. Bottom: Densitometric analysis with the abundance of total Fus3. For densitometric analysis, essentially the most intense band on every blot was set at 100 , along with the intensities of your other bands have been expressed as percentages of your maximum. Results are signifies SEM from 3 Cytochrome c/CYCS Protein Molecular Weight 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; 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. Information are expressed as percentages of your -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 have been 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 in 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 two 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; readily 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 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.
