FIGURE 1.

Individual amino acids have little effect on mTORC1 activity. A and B, HeLa cells starved in KRBH for 1 h were incubated in the absence (−) or in the presence of indicated amino acids for 40 min. Alternatively, starved cells were preincubated in the presence of Ser for 20 min followed by Leu for 20 min (S/L). A, the phosphorylation of S6K (Thr³⁸⁹), ULK1 (Ser⁷⁵⁷), S6 (Ser^240/244), and total S6K, ULK1, S6, and actin was analyzed by immunoblotting. B, the means of pS6K/S6K, pULK1/ULK1, and pS6/S6 are expressed as percentages compared with S/L-treated cells. The data shown are averages of two independent experiments (n = 4) ± S.D. Asterisks denote values significantly different from cells treated in the absence of amino acids. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (Tukey's test). C, starved cells were preincubated in the absence (−) or in the presence of Gln, Asn, Ser, or Val at 5 mm for 120 min (light bars) or at 20 mm for 30 min (dark bars) followed by incubation in the absence or in the presence of 5 mm Leu for another 20 min. The means of pS6K/S6K values from immunoblots are expressed as fold increase compared with starved cells. The data shown are averages of two independent experiments performed in duplicate (n = 4) ± S.D.