Figure 5.
Decreased mTORC1 activity in endocytosis-inhibited cells is rescued by overexpression of Rheb and constitutively active Rag. A, 293T cells were transfected with pRK5/HA-GST (mock); pRK5/HA-GST-RagB(99L) and pRK5/HA-GST(75L) (RagBGTP/RagCGDP); or pRK5/HA-GST-Rheb1 (Rheb). At 24 h post-transfection, cells were treated for 4 h with 0.1% DMSO (vehicle) or 80 μm Dynasore. B, quantification of immunoblot data in A. Each bar indicates mean ± S.D. of three independent experiments. **, p < 0.01. C, 293T cells were transfected as in A and subjected to immunofluorescence with anti-mTOR and anti-HA antibodies. Arrows indicate colocalization of mTOR and RagB/C. Scale bar = 10 μm. D, quantification of cells that exhibit punctate mTOR localization by analysis of image data in C. Only transfected (HA-positive) cells were examined. Each bar indicates mean ± S.D. of three independent experiments. ****, p < 0.0001.