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Figure S4. Tuberin mutants retain their ability to interact with hamartin. (A) Phosphorylation of tuberin does not alter tuberin-hamartin interaction. HEK293 cells were transfected with Flag-TSC2-WT and Myc-TSC1 constructs and were treated as described. Western blot analyses of immunocomplexes were performed to determine whether Myc-hamartin could be coimmunoprecipitated along with Flag-tuberin. (B) Flag-TSC2 constructs described in Fig. 3 A were overexpressed in HEK293 cells. Whole cell extracts were generated, and exogenous tuberin was immunoprecipitated using an anti-Flag antibody. Immunocomplexes were analyzed by Western blotting to detect Flag-tuberin and endogenous hamartin within the complex (top). As a control, the amounts of exogenous tuberin and endogenous hamartin within the whole cell extracts were also analyzed by Western blotting (bottom). (C) Flag-TSC2-WT or Flag-TSC2-2A constructs were overexpressed along with Myc-TSC1 in HEK293 cells. Whole cell lysates were generated and immunoprecipitations were performed with the indicated antibodies. Immunocomplexes were analyzed by Western blotting to determine whether Flag-tuberin and Myc-hamartin were present within the complex. The vertical line indicates nonadjacent lanes in a single blot. (D) MCF7 cells were transfected in serum-free media with wild-type Flag-TSC2 and Myc-TSC1 constructs. Localization of exogenous Flag-tuberin and Myc-hamartin was detected by double immunofluorescence labeling and confocal microscopy. Exogenous tuberin is shown in green, and exogenous hamartin is shown in red. Colocalized pattern of expression is shown as regions of punctate yellow signal. (E) HeLa cells were transfected in serum-free media with Flag-TSC2 2A mutant construct. After 12 h without serum, cells were treated with 20% FBS for 1 h. Localization of exogenous Flag-tuberin 2A mutant was detected by immunofluorescence labeling and fluorescence microscopy. Without the input of exogenous hamartin, the 2A mutant is predominantly punctate in the presence or absence of serum.