FIGURE 5.

Pik1 acts via Ste11 or a pathway component downstream of Ste11. A, TetO₇-PIK1 cells were transformed with pRS315-GAL1-STE4 and grown in selective medium containing dextrose or galactose to induce Ste4 (Gβ) protein expression. Cells were treated with 10 μg/ml doxycycline 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. B, TetO₇-PIK1 cells were transformed with pRS425 (Vector) or pRS425-STE11–4. C, P-Fus3 levels from A and B were quantified by scanning densitometry and analyzed with ImageJ software. Results are the mean ± S.E. for three individual experiments. D, TetO₇-STT4 cells treated as in A. E, TetO₇-STT4 cells treated as in B. F, P-Fus3 levels from D and E were quantified as in C.