Polyethyleneimine max mw 40 000

For transposon-mediated gene transfer, cells were transfected with the PiggyBac or Tol2 transposase expression vector (mPBase or pCAGGS-T2TP; Table S1) and the donor vectors using 293fectin (Thermo Fisher Scientific, Waltham, MA). One day after tge transfections, cells were selected by 10 μg/mL blasticidin S (InvivoGen, San Diego, CA) or 1.0 μg/mL puromycin (InvivoGen). For lentivirus-mediated gene transfer, HEK-293T cells were cotransfected with each pCSII vector, psPax2 (a gift from Didier Trono; Addgene plasmid # 12260), and pCMV-VSV-G-RSV-Rev (a gift from Dr. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan), by using polyethyleneimine "Max" MW 40,000 (Polyscience, Warrington, PA). Virus-containing media were collected 48 hours after transfection, filtered, and applied to target cells with 10 μg/mL polybrene (Nacalai Tesque). Two days after infection, the infected cells were selected with the following antibiotics: 200 μg/mL hygromycin (Wako, Osaka, Japan), 800 μg/mL G418 (InvivoGen), 10 μg/mL blasticidin S (InvivoGen), 1.0 μg/mL puromycin (InvivoGen), and 10 μg/mL zeocin (InvivoGen). Single-cell clones were isolated by the limiting dilution method.

E. coli C41(DE3)-RIPL cells were used for expression of all DEPTOR constructs as well as for 4EBP1, S6K1, RHEB and the isolated mTOR FRB domain. E. coli LOBSTR cells (EC1001, KeraFast) were used for the expression of PRAS40. The cells were grown in 2xTY media at 37 °C, induced at the OD600 = 0.7 with 0.3 mM isopropyl-d-1-thiogalactopyranoside, followed by 16 h of growth at 18 °C before harvest. Expi293F cells (Thermo Fisher A14527, RRID:CVCL_D615) were used for the production of mTORC1 and its mutants. Cells were grown in a Multitron Pro shaker set at 37 °C, 8% CO2 and 125 rpm. Cells were transfected at a cell density of 2.5x10 6 cells/mL by co-transfecting plasmids (1.1 mg total DNA/L cells) using PEI (Polyethyleneimine "MAX", MW 40,000, Polysciences, 24765, total 3 mg PEI/L cells).