Fig 3. Whi2 is a potent suppressor of TORC1 similar to Npr2 and Npr3.
(A) Immunoblot analysis detecting sustained TORC1 activity in mutant strains was assessed with anti-phospho-Rps6 before and after switching from control medium (SCCSH) to low (L) amino acids (SCME) for 3 h with/without addition of 20 ng/mL rapamycin for an additional 30 min (Rap). Equal loading was achieved primarily by optical density-matched cultures and monitored with anti-Pgk. Corresponding bars below show quantification of TORC1 activity (the ratio of phospho-Rps6/Pgk). Values were normalized to the average value of WT before switching media, and presented in the bar graph as means ± SD (n = 4 independent experiments). **P < 0.01; *P < 0.05; n.s., not significant, compared to the respective WT control using a two-tailed T test. (B) Growth of density-matched cultures spotted in parallel on control/high levels of amino acids (SCCSH) without or with 2.5 ng/mL rapamycin, and on low amino acid levels (SCME) without or with 2.5 ng/mL rapamycin. Representative of 3 independent experiments are shown. (C) Model depicting the position of Whi2 relative to the Iml1-Npr2-Npr3 and Gtr1-Gtr2 axis in the yeast TORC1 pathway. See text for explanation of numbered steps.