(A) Early–log phase cultures of WT and elm1Δsak1Δtos3Δ cells were left untreated or treated with 3 µM α-factor (α-F) for the indicated times before samples were harvested. Top: Western blotting analysis of samples with antibody against phosphorylated p44/42 MAPK (to detect p-Fus3 and p-Kss1), antibody against total Fus3 protein, and antibody against Gpa1. Glucose-6-phosphate dehydrogenase (G6PDH) was used as a loading control. Bottom: Densitometric analysis of the abundance of p-Fus3 in each sample normalized to the abundance of total Fus3 protein. Data are means ± SEM from three independent experiments. *P < 0.05. (B) Analysis of pheromone-dependent gene transcription in WT and elm1Δsak1Δtos3Δ cells. Cells expressing a FUS1-lacZ reporter were treated with the indicated concentrations of α-factor for 90 min, and then β-galactosidase activity was measured. Data are means ± SEM from three experiments, each performed in quadruplicate. Data are expressed as a percentage of the β-galactosidase activity of WT cells at the maximum concentration of pheromone. *P < 0.05. (C) Early–log phase cultures of WT and reg1Δ cells were left untreated or treated with 3 µM α-factor (α-F) for the indicated times before samples were harvested. Top: Western blotting analysis of samples with antibody against phosphorylated p44/42 (to detect p-Fus3 and p-Kss1), antibody against total Fus3 protein, and antibody against Gpa1. G6PDH was used as a loading control. Bottom: Densitometric analysis of the abundance of p-Fus3 in each sample normalized to the abundance of total Fus3 protein. Data are means ± SEM from three independent experiments. *P < 0.05. (D) Analysis of pheromone-dependent gene transcription in WT and reg1Δ cells. Cells expressing a FUS1-lacZ reporter were treated with the indicated concentrations of α-factor for 90 min, and then β-galactosidase activity was measured. Data are means ± SEM from three experiments, each performed in quadruplicate. Data are expressed as a percentage of the β-galactosidase activity of WT cells at the maximum concentration of pheromone. *P < 0.05.