(A) Silver stain of SDS-PAGE analysis of mTOR immunoprecipitates prepared from HEK-293E cells.
(B) Schematic representation of structural features of human DEPTOR and its mouse and chicken orthologues.
(C) Endogenous mTOR, raptor, and rictor co-immunoprecipitate with epitope-tagged DEPTOR. FLAG immunoprecipitates from HEK-293E cells expressing FLAG-DEPTOR, FLAG-PRAS40, or FLAG-tubulin were analyzed by SDS-PAGE followed by silver staining.
(D) Interaction of endogenous DEPTOR with endogenous mTORC1 and mTORC2 is sensitive to high salt-containing buffers. mTOR, raptor, rictor, or actin immunoprecipitates were prepared from HEK-293E cells, washed with buffers containing indicated amounts of NaCl and analyzed by SDS-PAGE followed by immunoblotting for indicated proteins.
(E) Endogenous DEPTOR co-immunoprecipitates endogenous mTOR. DEPTOR immunoprecipitates were prepared from HeLa cells, washed in a buffer containing 150 mM NaCl, and analyzed as in (D).
(F) DEPTOR interacts with mTOR and not mLST8/GβL. Indicated cDNAs in expression vectors were co-expressed in HEK-293T cells, cell lysates prepared, and used to prepare anti-HA immunoprecipitates and anti-myc immunoprecipitates. Both immunoprecipitates and cell lysates were analyzed as in (D).
(G) Myc-tagged mTOR, its indicated fragments, or rap2a were co-expressed in HEK-293T cells with FLAG-DEPTOR, and anti-myc immunoprecipitates were analyzed as in (D).
(H) The PDZ domain of DEPTOR interacts with mTOR. FLAG-tagged DEPTOR, its fragments, or metap2 were co-expressed in HEK-293T cells with myc-mTOR, and anti-FLAG immunoprecipitates were analyzed as in (D). Asterisks (*) indicate non-specific bands.
(I) Schematic indicating the regions of mTOR and DEPTOR that mediate their interaction. The abbreviations for and residues that define known mTOR domains are: HEAT Repeats, 1–1382; FAT, 1383–2014; R (FKBP12-Rapamycin Binding), 2015–2114; KD (PI3K-like Kinase Domain), 2115–2431; C (FATC), 2432–2549.