FIGURE 3.
Pik1 regulates pheromone signaling independently of Ste5. A, TetO7-STT4 cells were transformed with pRS313-GAL1-STE5-CTM and grown in selective medium containing dextrose or galactose to induce Ste5-CTM protein expression. Cells were treated with 10 μg/ml doxycycline (Dox) for 15 h and 3 μm α factor pheromone for 30 min, as indicated. Cell lysates were resolved by 12.5% SDS-PAGE and immunoblotting with phospho-p44/42 antibodies (P-Kss1 and P-Fus3) and G6PDH antibodies as a loading control. Phosphorylated and activated Fus3 (P-Fus3) was quantified by scanning densitometry and analyzed with ImageJ software. Results were normalized to P-Fus3 levels of untreated samples. B, TetO7-PIK1 cells treated as in A. Results are the mean ± S.E. for three individual experiments.