Fig. 5

Increased mTORC1 activity in cells with elevated Src activity. a FHC and Caco-2 cells were amino acid starved (kept in dialyzed serum) for 4 h prior to lysis and immunoblot analysis to measure the levels of the indicated proteins and phosphorylation states. b FHC and HT-29 cells were treated as in a prior to immunoblot analysis to measure the levels of the indicated proteins and phosphorylation states. Quantified data in a and b represent means ± SEM of n = 2–3 independent experiments. **p < 0.001. c FHC, Caco-2 and HT-29 cells were starved as in a and then stimulated with amino acids for 30 min prior to immunofluorescence labeling of endogenous LAMP2 (green) and mTOR (red). Bars indicate 40 μm in FHC cells and 80 μm in Caco-2 and HT-29 cells. Representative cells are shown where yellow or orange pixels indicate co-localization in the merged images. Insets show selected fields that were magnified by a factor of 6. Quantified data represent co-localization (Mander’s coefficient) means of mTOR and LAMP2 in at least 15 cells for each condition. *p < 0.05 and **p < 0.01. d Extent of co-localization of mTOR and LAMP2 in FHC, Caco-2 and HT-29 cells under amino acid starvation. Box plots in c and d represent quantified data of co-localization (Mander’s coefficient) analysis in at least n = 30 cells/line. ***p < 0.001. e Caco-2 cells were amino acid starved (kept in dialyzed serum) for 4 h and treated with PP2 for the last 2 h prior to 30-min amino acid stimulation. Cells were lysed and immunoblot analyses were performed to measure the levels of the indicated proteins and phosphorylation states. Statistical differences between groups in a–d were determined using ANOVA with Tukey’s post hoc test