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 with the indicated concentrations of -factor for 90 min, after which -galactosidase activity was measured. ALDH1 list Information are means SEM from 3 experiments, each performed in quadruplicate. Information are expressed as a percentage of your -galactosidase activity of WT cells at the maximum concentration of pheromone. P 0.05.NIH-PA Author Caspase 3 Formulation Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; out there in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 4. Crosstalk involving mating and glucose-sensing pathways(A to C) Analysis from the effects of higher and low glucose on the abundance and phosphorylation of Fus3. (A) WT cells, (B) elm1sak1tos3 cells, and (C) reg1 cells had been cultured in medium containing 2 or 0.05 glucose for 5 min just before getting left untreated or treated with 3 -factor (-F) for the indicated occasions prior to they had been harvested for analysis. Major: Samples had been analyzed by Western blotting with antibody against phosphorylated p4442 MAPK (to detect p-Fus3 and p-Kss1), too as with antibodies specific for total Fus3, Gpa1, phosphorylated Snf1 (p-Snf1), and G6PDH, which was utilised as a loading manage. Middle: Densitometric evaluation on the abundance of p-Fus3. Bottom: Densitometric analysis in the abundance of total Fus3. For densitometric analysis, probably the most intense band on every blot was set at one hundred , and the intensities from the other bands have been expressed as percentages of 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 were left untreatedSci Signal. Author manuscript; readily available 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 2 glucose, which was set at 100 . Information are indicates SEM from three independent experiments, each and every 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 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 3 -factor for five min, whereas cells expressing STE11-4 have been collected 5 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 evaluation from the intensities of bands corresponding to p-Fus3, normalized to these corresponding to total Fus3. For each set of cells, the abundance of p-Fus3 in 2 glucose was set at 100 . Information are indicates 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 situations of 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.
