FIGURE 6:

Early and intermediate endocytic mutants have no effects on TORC2 signaling. (A) Wild-type and ede1∆ cells were grown at 30°C to log phase in YPD medium, and Ypk-pT662 and Ypk1 protein were assayed by Western blot. Quantification of Ypk-T662 phosphorylation and total Ypk1 protein are shown. (B) Wild-type and ede1∆ cells were grown in YPD medium to early log phase and were then rapidly washed into YPG/E medium at 30°C. Cells were collected at the indicated time intervals, and levels of Ypk-pT662 and Ypk1 protein were assayed by Western blot. An asterisk indicates a background band that overlaps with the Ypk1 signal. (C) Cells of the indicated genotypes were grown at 30°C to log phase in YPD medium. Auxin (1 mM) was added, and cells were collected and analyzed by Western blot at the indicated time points. Quantification of Ypk-T662 phosphorylation and total Ypk1 protein are shown. An asterisk indicates a background band that overlaps with the Ypk1 signal. (D) Cells of the indicated genotypes were grown in YPD medium to early log phase. Auxin (1 mM) was added, and after 30 min cells were washed into YPG/E medium at 30°C containing 1 mM auxin. Cells were collected at the indicated time intervals, and Ypk-pT662 and Ypk1 were assayed by Western blot. An asterisk indicates a background band that overlaps with the Ypk1 signal. Error bars represent the SD of the mean of three biological replicates. *** indicates a p value smaller than 0.005 and * indicates a p value between 0.05 and 0.01 when the indicated strain is compared with the wild type .