Fig 1. Cells lacking Tor1 N-terminal HEAT repeats were rapamycin hypersensitive.
A. Cells expressing TOR1-Del381 or TOR1-FL from tetO were pre-grown in YPD medium for 4 h and inoculated in YPD with increasing concentrations of doxycycline (Doxy). OD600 was read every 15 minutes. B. Western blot of cells of indicated TOR1 genotypes, wild type (TOR1/TOR1), Del381 and FL, grown in YPD with 5 ng/ml doxycycline for 3.5 h, then inoculated into YPD with 5 ng/ml doxycycline and incubated for indicated time periods (15 min, 30min, 45min, 60min); protein extracts probed with antibody to phosphorylated Rps6 (P-S6), total Rps6 (S6) and tubulin (Tub) as loading control. Dens: signal intensity ratio of P-S6 to Tub. (TOR1/TOR1, JKC1713; tetO-TOR1-Del381, JKC1441; tetO-TOR1, JKC1549). The same samples were run on separate gels to detect either P-S6 or S6. C. Dilutions of cells of indicated genotypes were spotted on YPD medium containing vehicle (Veh, 90% ethanol) or 1.5 ng/ml rapamycin (Rapa), without or with 300 ng/ml doxycycline and incubated at 30o for 2 days. (TOR1/TOR1, JKC1713; tor1/TOR1, JKC1345, JKC1346, JKC1347; tetO-TOR1-Del381, JKC1442, JKC1445, JKC1441; tetO-TOR1, JKC1543, JKC1546, JKC1549). D. Cells of indicated genotypes were grown in YPD medium containing Vehicle (Veh, 90% ethanol), 1.5 ng/ml Rapamycin (Rapa) or 1.5 mM Caffeine (Caff), without or with 300 ng/ml doxycycline. (TOR1/TOR1, JKC1713; tor1/TOR1, JKC1347; tetO-TOR1-Del381, JKC1441; tetO-TOR1, JKC1549). E. Cells of indicated genotypes were pre-grown in YPD medium with 5 ng/ml doxycycline for 3.5 h (Time 0) and then diluted into fresh YPD medium with 5 ng/ml doxycycline and 25 ng/ml rapamycin. Strains, antibodies and densitometry as in B.