Phos-tag western blot analysis of (A) wild-type cells or (B)
bar1Δ cells treated with 0.3 μM or 3
μM α-factor mating pheromone and probed with anti-Myc tag
antibodies to identify dually phosphorylated (pp-Kss1), mono-phosphorylated
(p-Kss1), and non-phosphorylated (Kss1) Kss1. pp-Kss1, p-Kss1, and np-Kss1
(first and second graphs) are plotted as % of lane total. Total Kss1 (third
graphs) is plotted as % maximum lane signal on the blot. The ratio of dually
phosphorylated Kss1 to total Kss1 (bottom graphs) was calculated by dividing
the % dually phosphorylated Kss1 in each lane by % total Kss1 in each lane.
Data are presented as mean ± standard deviation, N = 3.