[HELP with High Resolution Image Viewing][To Figure]

[View Larger Version of this Image]

Figure S1. AKT-mediated phosphorylation of tuberin leads to subcellular translocation. (A)Serum stimulation increases the ratio of cytosolic tuberin/membrane tuberin. NIH3T3 fibroblasts were serum starved (SV) and then serum stimulated (SM) for 1 h, and cells were fractionated. Lysates were analyzed by Western blot, and the ratio of cytosolic tuberin and membrane tuberin protein levels was quantified. Serum stimulation led to an approximately fourfold higher ratio of cytosolic tuberin/membrane tuberin when compared to starved NIH3T3 cell lysates (top). The vertical line indicates nonadjacent lanes in a single blot. An anti-phospho-AKT antibody (S473) was used to show that serum stimulation activated the AKT signaling pathway (middle). (B)PI3K inhibitors block tuberin translocation to the cytosol. Western blot analyses of tuberin levels within membrane and cytosolic fractions of TRKE2 and MCF7 cells that were stimulated with serum (SM) or 30 ng/ml IGF-1 in the absence or presence of PI3K inhibitors, 200 nM wortmannin, or 20 µM LY294002 (LY). Graphs representing the ratio of cytosolic tuberin to membrane tuberin are shown below each Western blot. (C) S1338A and Δ73 mutant tuberin do not exhibit altered localization. HEK293 cells were transfected with the indicated Flag-TSC2 constructs, which were described in Fig. 3 A. Cells were fractionated, and exogenous tuberin was detected by Western blot analysis.