Fig. 6. MET mediated MTOR activation through V-ATPase, not AKT.

a, b MET is required for amino acid-stimulated MTOR activation. WT and MET-KO HepG2 (a) and H22 (b) cells were individually deprived of amino acids for 90 min and then stimulated with or without amino acids for 45 min. Cell lysates were subjected to immunoblotting with the indicated antibodies. c MET participates in amino acid-stimulated MTOR activation in an AKT-independent manner. WT and MET-KO HepG2 cells were individually treated with or without 1 μM afuresertib for 12 h, deprived of amino acids for 90 min, and subsequently stimulated with amino acids for 45 min. Cell lysates were subjected to immunoblotting with the indicated antibodies. d MET functions upstream of the Rag GTPase complex upon MTOR signaling. WT and MET-KO HepG2 cells were individually transfected with or without the RagB Q99L (RagB^GTP) mutant for 36 h, deprived of amino acids for 90 min, and subsequently stimulated with amino acids for 45 min. Cell lysates were subjected to immunoblotting with the indicated antibodies. e, f MET controlled amino acid-stimulated MTOR activation via V-ATPase. MET-KO HepG2 (e) and HEK293T (f) cells were individually transfected with Flag-MET or a vector control for 36 h, starved for 90 min in the presence of 5 μM concanamycin A (Con A) or a vehicle, and subsequently stimulated with amino acids for 45 min. Cell lysates were subjected to immunoblotting with the indicated antibodies.