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 have been treated together with the indicated concentrations of -factor for 90 min, and then -galactosidase activity was measured. Data are implies 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 Kinesin-7/CENP-E supplier 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) Analysis in 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. Top: Samples have been 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 employed as a loading handle. Middle: Densitometric analysis in the abundance of p-Fus3. Bottom: Densitometric evaluation of your abundance of total Fus3. For densitometric analysis, essentially the most intense band on every blot was set at 100 , as well as the intensities of your other bands have been 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; accessible 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 with the -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 5-HT5 Receptor custom synthesis plasmid or with plasmid encoding STE11-4, a constitutively active mutant in the MAPKKK Ste11. Early og phase cells had been resuspended in medium containing either 2 or 0.05 glucose. Cells transformed with empty plasmid have been treated with three -factor for 5 min, whereas cells expressing STE11-4 had been 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 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 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) had been grown in medium containing two glucose. Cells (1 107) f.
