Fig. 3

Src promotes mTORC1 recruitment to lysosomes. a HEK cells transiently transfected with scrambled shRNA or shSrc prior to coIP analyses. Lysates were used for immunoprecipitation by either Src or IgG antibody (negative control). b, c HEK293 cells were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). coIP analyses were performed to test interaction of mTOR and Raptor with RagA in b or RagB in c. Immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states. d HEK293 cells stably expressing Flag-raptor, transiently transfected with HA-GST-RagA^WT/C^WT or HA-GST-RagA^GTP/C^GDP, were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). coIP analyses were performed to test interaction of mTOR and Raptor with RagA/C. Immunoblot analyses were used to measure the levels of the indicated proteins. e, f HEK293 cells were transiently transfected with HA-GST-RagA^WT/C^WT (e) or HA-GST-RagA^GTP/C^GDP (f) and treated as in a prior to immunofluorescence labeling of HA (green) and endogenous mTOR (red). Representative cells are shown where punctate structures indicate lysosomal localization of mTOR. Magnified images are represented in white boxes. Bar indicates 40 μm. g HEK293 cells stably expressing HA-GST-RagA^WT/C^WT or HA-GST-RagA^GTP/C^GDP were treated as in b. Immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states. Short and long indicate short and long exposure, respectively. Box plots represent SE of n = 3 independent experiments. ***p < 0.001. Statistical differences between groups were determined using ANOVA with Tukey’s post hoc test. GAPDH was used as a loading control in all immunoblot assays