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. 2021 May 11;12:2695. doi: 10.1038/s41467-021-22908-4

Fig. 4. Tanc2 interacts with mTOR.

Fig. 4

a Purified Tanc2 protein directly interacts with purified mTOR protein. GST-Tanc2 (full-length) protein was used to pull down purified mTOR protein. Input, 20%. Three independent experiments yielded similar results. b, c Tanc1 and Tanc2 form a complex with mTOR in the mouse brain. Whole-brain lysates (P14; mouse) were immunoprecipitated (IP) with pan-Tanc or mTOR antibodies, followed by immunoblotting. Note that mTOR pull-down also coprecipitated PSD-95 through Tanc2. Three independent experiments yielded similar results. d Both N- and C-terminal regions of purified Tanc2 protein directly interact with purified mTOR but not purified Deptor. GST-tagged purified N- and C-terminal regions of human Tanc2 (aa 1-1358 and aa 1359-1990) were coupled to glutathione beads and incubated with purified mTOR and Deptor proteins, followed by GST pull-down and immunoblot analysis. Three independent experiments yielded similar results. e Tanc2 forms a complex with mTOR in HEK293T cells through multiple regions of Tanc2. Lysates of HEK293T cells expressing deletion variants of Flag-Tanc2 (near-full-length, aa 127-1990; N-terminal region, aa 127-835; middle region, aa 836-1358; C-terminal region, aa 1234-1990) and mTOR (endogenous) were immunoprecipitated with Flag antibodies and immunoblotted with anti-Flag (for Tanc2) and mTOR antibodies. Note that all four deletion variants of Tanc2 interacted with mTOR, suggesting that multiple regions of Tanc2 are involved in mTOR binding. We used the near-full-length Tanc2 because the full-length construct was unavailable at the time of the experiment; experiments repeated using the full-length Tanc2 construct yielded the same results. ANK ankyrin repeats, TPR tetratricopeptide repeats, CC coiled-coil domain, PB PDZ-binding motif. Three independent experiments yielded similar results. f The C-terminal region of mTOR containing FRB, kinase, and FATC domains is sufficient for complex formation with Tanc2. HEK293T cells expressing Myc-mTOR deletion variants and Flag-Tanc2 (aa 127-1990) were immunoprecipitated with Flag antibodies and immunoblotted with Myc and Flag antibodies. FRB, FKBP12-rapamycin binding; FATC, FRAP-ATM-TRRAP-C-terminal domain. Three independent experiments yielded similar results.