Fig. 2: Increased mTOR activity in Tanc2-mutant mice. | Nature Communications

Fig. 2: Increased mTOR activity in Tanc2-mutant mice.

From: Tanc2-mediated mTOR inhibition balances mTORC1/2 signaling in the developing mouse brain and human neurons

Fig. 2

a Increased mTOR activity (mTORC1 and mTORC2) in the brain of Tanc2+/− (HT) mice (P14; male), as shown by Ser-2448 phosphorylation. Note that phosphorylation levels of 4E-BP1 (Thr-37/46), but not S6 (Ser-235/236), downstream of mTOR were also increased. Note also that the increased phosphorylation of Akt (S473) indicates mTOR activation in the mTORC2 complex. For quantification, HT and WT signals were normalized to those of α-tubulin. See Source Data 2 for uncropped immunoblot images for this panel and all other immunoblot panels. Data: mean ± SD (standard deviation). Numbers on the left side of the immunoblots indicate molecular masses in kDa. (n = 4–8 mice [WT, HT], except for 8 for mTOR, p-mTOR, and Tanc1/2, *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). b Lack of changes in phosphorylated or total levels of PI3K, PTEN, and TSC2 proteins upstream of mTOR in the Tanc2+/− brain (P14; male). Immunoblot signals were normalized to α-tubulin signals. Data: mean ± SD. (n = 4 mice [WT, HT], Student’s t test). c Normal phosphorylated and total levels of mTOR (S2448), S6 (S235/236), 4E-BP (T37/46), Akt (S473), and GSK3β (S9) in the Tanc2+/− brain (P28; male). Immunoblot signals were normalized to α-tubulin signals. Data: mean ± SD. (n = 4 animals [WT, HT], ***P < 0.001, Student’s t test). d Normal phosphorylated and total levels of mTOR (S2448), S6 (S235/236), 4E-BP (T37/46), Akt (S473), and GSK3β (S9) in the Tanc2+/− brain (P52; male). Immunoblot signals were normalized to α-tubulin signals. Note that 4E-BP phosphorylation is moderately decreased. Data: mean ± SD. (n = 6 animals [WT, HT], **P < 0.01, ***P < 0.001, Student’s t test). e, f Cre-dependent acute hippocampal Tanc2 deletion during P5–14 leads to increased phosphorylation of S6 (S235/236), 4E-BP (T37/46), Akt (S473), GSK3β (S9), and mTOR (S2448), indicative of mTORC1 and mTORC2 hyperactivity. AAV1-Synapsin-Cre-eGFP and AAV1-Synapsin-eGFP (control) were injected in parallel into both sides of the hippocampus (CA3 region) of Tanc2fl/fl mice (eGFP expression is indicated in green). Note that S6 phosphorylation was increased, in contrast to the normal S6 phosphorylation observed in the Tanc2+/− brain (Fig. 2a). Data: mean ± SD. (n = 6 independent experiments, *P < 0.05, ***P < 0.001, Student’s t test). g, h Cre-dependent acute hippocampal (CA3 region) Tanc2 deletion during P19–28 does not increase phosphorylation of S6 (S235/236), 4E-BP (T37/46), Akt (S473), GSK3β (S9), or mTOR (S2448), indicative of the lack of mTORC1 and mTORC2 hyperactivity. Note that total (not phosphorylation) levels of S6 were increased, and that total and phosphorylation levels of 4E-BP were decreased (not increased). Data: mean ± SD. (n = 6 independent experiments, ***P < 0.001, Student’s t test). See Source Data 1 for raw data values and Supplementary Table 1 for statistical details.

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