Fig 1: Tanc2 inhibits mTOR activity.a Tanc2 suppresses endogenous mTOR activity in HEK293T cells. HEK293T cells were transfected with near-full-length Flag-Tanc2 (aa 127-1990; human) or Flag-Tanc2 deletion variants (aa 836-1986 and 1234-1990; human), followed by immunoblot analysis for phosphorylated (Ser-2448) and total mTOR. Cells in which mTOR was suppressed by rapamycin served as controls. Note that the near-full-length variant significantly inhibited mTOR activity, whereas the shorter deletion variants (aa 836-1986, aa 1234-1990) did not. Data: mean ± SD. (n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 [compared with the first bar in each graph], one-way ANOVA with Bonferroni test). b Purified Tanc2 inhibits the kinase activity of purified mTORC1 (containing GFP-mTOR and mTOR-associated proteins such as Raptor), as shown by decreased phosphorylation of purified S6K (an mTOR substrate). In control experiments, EGFP-Tanc2 was replaced with purified EGFP protein (lanes 1–4; not probed). Data: mean ± SD. (n = 3 independent experiments, *P < 0.05 [compared to the absence of Tanc2/the second or fourth bar in the middle and right panels, respectively], ns not significant, one-way ANOVA with Bonferroni test). c Purified Tanc2 inhibits the kinase activity of purified mTORC2 (containing GFP-mTOR- and mTOR-associated proteins such as Rictor), as shown by decreased phosphorylation of purified Akt (an mTOR substrate). The baseline phosphorylation in Akt could be attributable to that a small portion of purified Akt proteins is phosphorylated or that the antibody recognizes non-phosphorylated proteins in addition to phosphorylated proteins. Data: mean ± SD. (n = 3 independent experiments, ***P < 0.001 [compared to the absence of Tanc2/the fourth bar], ns not significant, one-way ANOVA with Bonferroni test). See Source Data 1 for raw data values and Source Data 2 for statistical details.