Figure 4. Distribution of components of the mTORC1 complex in endomembranes and cytosol.

Membranes were separated from cytosol as in (5) in HCT116 cells (A) or were fractionated as in (6) for isolation of heavy membranes containing lysosomal membrane (B, C, D) from light membranes and cytosol, and immunoblotted for components of the mTORC1 complex. (D) Fractionated membranes from MEFs wt or null for TSC2. TSC2 −/− MEFs can only be made in a p53 −/− background. LDH was used as a cytosolic marker, Integrin β1 for membranes, and LAMP1 for lysosomal membranes (The LAMP1 antibody does not detect the mouse epitope in D). The bar graphs represent data from 3 experiments (mean ± SEM). TSC2 in heavy membrane fractions was significantly lower in p53 null cells ((B) * denotes p= 0.004) and Rheb was significantly higher compared to p53 wt HCT116 ((C) p= 0.002). There were significantly higher Rheb levels in heavy membranes from TSC2 null MEFs ((D) p= 0.007).