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. 2022 Jun 10;18(6):e1010089. doi: 10.1371/journal.ppat.1010089

Fig 3. Cells lacking Tor1 N-terminal HEAT repeats failed to adapt growth to carbon source availability and were defective in growth on non-fermentable carbon sources.

Fig 3

A. Cells were grown in YNB medium with varying concentration of glucose (Glu), without or with 300 ng/ml doxycycline (300 Doxy). B. Cells of indicated genotypes were spotted onto YNB without inositol agar medium containing different carbon sources (2% w/v), without or with 300 ng/ml doxycycline. (TOR1/TOR1, JKC1713; tor1/TOR1, JKC1345, JKC1346, JKC1347; tetO-TOR1-Del381, JKC1442, JKC1445, JKC1441; tetO-TOR1, JKC1543, JKC1546, JKC1549). C. Cells of indicated genotypes were spotted onto YPD agar medium without or with 300 ng/ml doxycycline and incubated either aerobically or anaerobically for 48 h. D. Western blot. Cells pre-grown in YPD with 5 ng/ml doxycycline for 4 h were inoculated into YNB with 5 ng/ml doxycycline containing final nutrient concentrations as indicated (2% Glu: 2% glucose, 10 mM Pi; 0.25% Glu: 0.25% glucose, 10 mM Pi; 0% Glu: no added direct carbon source, 10 mM Pi; 2% Gly: 2% glycerol, 10 mM Pi; 0.05 mM Pi: 2% glucose, 0.05 mM Pi). Protein extracts were probed with antibody to phosphorylated Rps6 (P-S6) or phosphorylated eukaryotic translation initiation factor 2A (P-eIF2a), and tubulin (Tub) as loading control. Dens: signal intensity ratio of P-S6 or P-eIF2a to Tub. (TOR1/TOR1, JKC1713; tor1/TOR1, JKC1346; tetO-TOR1-Del381, JKC1445; tetO-TOR1, JKC1546). Same Western blotting membranes were sequentially probed with P-S6 antibody and then P-eIF2a antibody.