cDNA for S6K1 and 4EBP1-GFPSpark constructs were obtained from Sino Biological (China); mCherry-raptor, YFP-mTOR, YFP-PRAS40 and FLAG-mTOR from Addgene (USA). EGFP-mTOR, mDsRed-Rheb, EGFP-Rheb and mDsRed-raptor constructs were cloned previously21 (link). Miniprep and Maxiprep kits purchased from Qiagen (Germany). Rapamycin, L-leucine, L-serine and Ponceau S stain were purchased from Sigma-Aldrich (USA).
HEK293 cell line was purchased from ATCC® (USA), tested for mycoplasma contamination, and HEK293F cells were provided by Evotec (UK) Ltd. Cell culture dishes (35 mm × 20 mm) were bought from MatTek (USA).
HEK293 and HEK293T cells were cultured in Minimum Essential Media (MEM) supplemented with 10% Foetal Bovine Serum (FBS), 2 mM L-glutamine and 1% Penicillin-Streptomycin (P/S) for HEK293. HEK293F cells were grown in serum-free media (Gibco/Life Technologies, UK). All culture reagents were from Life Technologies. Cells were incubated at 37 °C with 5% CO2 humidified air in T75 culture flasks (Thermo Fisher Scientific, UK) or in 50 mL tubes (Corning®). SF9 cells were cultured in SF 900 III media supplemented with 1% P/S at 26 °C.
HEK293 cell line was purchased from ATCC® (USA), tested for mycoplasma contamination, and HEK293F cells were provided by Evotec (UK) Ltd. Cell culture dishes (35 mm × 20 mm) were bought from MatTek (USA).
HEK293 and HEK293T cells were cultured in Minimum Essential Media (MEM) supplemented with 10% Foetal Bovine Serum (FBS), 2 mM L-glutamine and 1% Penicillin-Streptomycin (P/S) for HEK293. HEK293F cells were grown in serum-free media (Gibco/Life Technologies, UK). All culture reagents were from Life Technologies. Cells were incubated at 37 °C with 5% CO2 humidified air in T75 culture flasks (Thermo Fisher Scientific, UK) or in 50 mL tubes (Corning®). SF9 cells were cultured in SF 900 III media supplemented with 1% P/S at 26 °C.
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