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. 2014 Dec 1;25(24):3954–3961. doi: 10.1091/mbc.E14-01-0004

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© 2014 Zeng et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — LanCL2 binds mTOR and facilitates Akt phosphorylation by mTORC2. (A) HepG2 cells were transfected with FLAG-tagged LanCL2, and FLAG IP was carried out 48 h after transfection. (B and C) HepG2 cells were infected with lentiviruses expressing a control shRNA (“C”), rictor shRNA (“ric”), or mTOR shRNA (“M”) for 2 d; this was followed by puromycin selection for 2 d. Cells were then transfected with FLAG-tagged LanCL2, and FLAG IP was carried out 48 h after transfection. (D) HepG2 cells were infected with lentiviruses expressing control shRNA or LanCL2 shRNA (“L”) and selected by puromycin. Cells were then transfected with HA-Akt, and HA IP was carried out 48 h after transfection. (E) Endogenous raptor and rictor were immunoprecipitated from HepG2 cells and subjected to mTORC1 and mTORC2 kinase assays, using GST-S6K1 and His-Akt as substrates, respectively. Phosphorylation at Thr-389-S6K1 or Ser-473-Akt was detected as a readout of the kinase activity. Purified His-LanCL2 (5 μg) was added before the kinase assay in the indicated samples. (F) HEK293 cells were transfected with FLAG-LanCL2 or an empty vector; this was followed by FLAG IP and Western analysis.