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 all the indicated concentrations of -factor for 90 min, after which -galactosidase activity was measured. Data are indicates SEM from three experiments, every single performed in quadruplicate. Data are expressed as a percentage with 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 Epiregulin Protein custom synthesis Signal. Author manuscript; accessible in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. four. Crosstalk in between mating and glucose-sensing pathways(A to C) Analysis of 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 just before getting left untreated or treated with 3 -factor (-F) for the indicated instances ahead of they have been harvested for analysis. Top: Samples had 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 used as a loading control. Middle: Densitometric analysis with the abundance of p-Fus3. Bottom: Densitometric analysis in the abundance of total Fus3. For densitometric evaluation, the most intense band on each blot was set at one hundred , as well as the intensities on the other bands had been expressed as percentages on the maximum. Final 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; 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. Information are expressed as percentages on the -galactosidase activity of pheromone-treated WT cells cultured in two glucose, which was set at 100 . Information are indicates SEM from three independent experiments, every single performed in quadruplicate. P 0.05. (E) WT cells had been 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 2 or 0.05 glucose. Cells transformed with empty plasmid had been treated with three -factor for 5 min, whereas cells expressing STE11-4 had been collected five min right after resuspension in fresh medium. Samples were analyzed by Western blotting with antibodies against phosphorylated p4442 MAPK and total Fus3. Bar graphs represent densitometric evaluation 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 one hundred . Data are implies 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 below situations of IL-1 alpha Protein medchemexpress restricted 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.
