(A) Schematic illustration of cell collection. Cells were grown at 30°C until exponential phase, washed once with medium lacking glucose and re-suspended in medium lacking glucose. Glucose was then added to the culture to a final concentration of 2%. Cells were collected at indicated times. (B) Wild-type cells and pho85Δ mutant cells harboring YCplac33-POP2FLAG were collected as in (A). Extracts prepared from each strain were run on phos-tag and conventional gels, then immunoblotted with anti-Flag antibody. Phosphorylated Pop2Flag is indicated with arrows and numbers according to the positions. Representative data are shown. (C, D, E) The intensities of Pop2Flag shifted-band 1, 3, and 5 signals in phos-tag gel were measured and normalized to the Pop2Flag signals in normal gel.