Cell Culture, Vectors and Transfection
HEK293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% (v/v) CO2. Firefly Luciferase (FLuc) andGaussia Luciferase (GLuc) expressing Chinese Hamster Ovary (CHO) cells were cultured in Ham’s F12 medium containing 10% foetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% (v/v) CO2. EXPI-CHO cells were acquired from ThermoFisher Scientific (SA, Australia) and cultured in Expi-CHO™ Expression Medium. All cells were regularly tested for mycoplasma contamination. pRK7-FLAG-Rheb vectors were used as previously described (Jianling Xie et al., 2020). peGFP-N1 neomycin selection vector was a kind gift from Dr. Timothy Sargeant. To create stable transfection vectors, DNA sequence encoding FLAG-Rheb was amplified from pRK7-FLAG-Rheb vectors via PCR with the addition of 5’ EcoRI and 3’ BamHIrestriction sites. Amplicons were cloned into peGFP-N1 neomycin selection vector via the EcoRI and BamHI restriction sites. Transfection into HEK293, FLuc-CHO and GLuc-CHO was performed using Lipofectamine 3000 (ThermoFisher Scientific, SA, Australia) according to the manufacturer’s instructions. Transfections into EXPI-CHO™ was performed via the Expifectamine™ transfection system as per the manufacturer’s instructions.