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Figure S2. S939 and S981 are required for altered subcellular localization of tuberin. (A) S1338A and Δ73 mutant tuberin translocate from membrane (M) to cytosol (C) in response to serum stimulation, similar to wild-type tuberin. HEK293 cells were transfected with the indicated Flag-TSC2 constructs for 24 h. Cells were then serum starved (SV) for 24 h and treated with or without 20% serum (SM) for 1 h as indicated. Cells were fractionated, and exogenous tuberin was detected by Western blot analysis. Phos-AKT (S473) was blotted as a control for the effectiveness of the serum stimulation. (B) Overexpression of RSK does not alter localization of tuberin. HEK293 cells were transfected with Flag-TSC2-WT along with an HA-RSK construct. Cells were fractionated and immunoblotted to detect exogenous tuberin and phosphorylated RSK. For comparison, exogenous tuberin-2A was also analyzed by Western blotting. β1-integrin and LDH proteins were used as fractionation controls. The vertical line indicates nonadjacent lanes in a single blot. (C) Phosphorylation of both S939 and S981 is required for tuberin to localize to the cytosol. HEK293 cells were transfected with the indicated Flag-TSC2 constructs, which were described in Fig. 3 A. Whole cell extracts (WC) and fractionated lysates were generated from transfected cells, and phospho-tuberin (p939) was detected by Western blot analysis. β1-integrin and LDH proteins were used as fractionation controls. Phospho-tuberin (p939) could be detected in both the membrane and cytosolic fractions when overexpressed Flag-tuberin-WT was analyzed; however, cytosolic phospho-tuberin (p939) could not be detected when the Flag-tuberin-S981A construct was overexpressed, indicating that S981 is necessary for phospho-tuberin (p939) to be detected within the cytosol. A schematic diagram of the results is shown below.