Figure 2. Ragulator-Rag complex responds to lysosomal damage in control of mTOR.

(A) mTOR activity (immunoblot analysis of S6K1 (T389) phosphorylation) in TSC2-deleted (TSC2^-/-) and wild type (TSC2^WT) cells treated with 100 μM GPN in full medium (Full) or starved in EBSS for 1 h. Ctrl (control): untreated cells. (B) Co-immunoprecipitation analysis of changes in interactions between Ragulator and Rag GTPases following treatment with GPN. HEK293T cells stably expressing FLAG-metap2 (control) or FLAG-p14 were treated with 100 μM GPN in full medium for 1 h. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted for endogenous RagA or RagC. (C) Immunoprecipitation (IP) analysis of interactions between RagB and mTOR/Raptor in cells treated with GPN. HEK293T cells overexpressing FLAG-metap2 (control) or FLAG-RagB were treated with 100 μM GPN in full medium for 1 h. Cell lysates were IP-ed with anti-FLAG antibody and immunoblotted for endogenous mTOR or Raptor. (D) mTOR activity in HEK293T cells or HEK293T cells stably expressing constitutively active RagB GTPase (RagB^Q99L) treated and analyzed as in A. (E) Immunofluorescence confocal microscopy visualization of mTOR localization relative to LAMP2-positive lysosomes. Cells as in D were treated as in A, and immunostained for endogenous LAMP2 (green florescence, Alexa-488) and mTOR (red florescence, Alexa-568). Scale bar, 1 μm. (F) Quantification by HC of overlaps between mTOR and LAMP2 (images, Figure S2C) in cells as in D, treated as in A. Data, means □ SEM; immunoblots: n ≥ 3; HC: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥ 5 wells/sample). † p ≥ 0.05 (not significant), *p < 0.05, **p < 0.01, ANOVA. See also Figure S2.