Protrudin is required for full mTORC1 activation after nutrient starvation–recovery. (A and B) RPE-1 (A) or HEK293 (B) cells grown in complete medium were transfected with control RNA or siRNA targeting Protrudin, starved in EBSS for 4 h, and stimulated or not with 1× amino acids (AA) in DMEM with or without growth factors for 2 h, before immunoblotting with the antibodies indicated. Graphs represent the relative levels of p-ULK1 or p-S6K. Error bars denote ± SEM from independent experiments. RPE-1: p-S6K (n = 3), p-ULK1 (n = 4). HEK293: p-S6K (n = 5), p-ULK1 (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001. No treatment (one-sample t test), samples with equal variance (unpaired t test). (C) RPE-1 cells were transfected with control RNA or siRNA targeting Protrudin. The cells were serum starved for 4 h and stimulated with FCS, insulin, and 1× amino acids in DMEM for 15 min and analyzed by immunoblotting. Error bars denote ± SEM from independent experiments (n = 3). *, P < 0.05 (one-sample t test). (D) RPE-1 cells were treated as in C, stained with antibodies against LAMP1 and p-AKT(Thr308), and analyzed by confocal microscopy.