Bromodeoxyuridine (BrdU) Assay
Was performed as previously described (Jianling Xie et al., 2020). BrdU Cell Proliferation Assay Kit (Cell Signalling Technology, Danvers, MA #6813) was performed as per the manufacturer’s instructions. Cells were plated at 10,000 cells per well in a 96-well plate 2 h prior to addition of 10 µl of 10x BrdU solution. 2 h after addition of BrdU, cells were fixed/denatured for 30 min. BrdU detection antibody was diluted 1:100 and added to cells for 1 h. Cells were washed 3x in wash buffer before addition of HRP-linked secondary antibody for 30 min. Cells were washed 3x before addition of 3,3’,5,5’-tetramethylbenzidine. After 30 min, STOP solution was added and absorbance determined at 450 nm.
Firefly and Gaussia Luciferase Assays
Firefly luciferase assay was performed using Steady-Glo® Luciferase Assay System (Promega, NSW Sydney) as per the manufacturer’s instructions. To prepare cells, FLuc-CHO cells were seeded in 6-well plates and transfected with vector encoding Rheb mutants. Cells were grown for 24 h and then seeded into 96-well plates. Cells were allowed to grow for 24 h before Firefly Luciferase assay was performed. 100 µL of assay reagent was added to each well and 5 min allowed for cell lysis. Luminescence was then measured using GloMax® Discover microplate reader (Promega Australia, NSW, Australia)
Gaussia luciferase (GLuc) assays were performed using the BioLux®Gaussia Luciferase Assay Kit (New England Biolabs, Ipswich, MA) as per the manufacturer’s instructions. Cells were prepared as per the Firefly luciferase assay. GLuc Assay solution was prepared by mixing 1:1000 BioLux® GLuc Substrate and BioLux® GLuc Assay buffer. 20 µL of cell growth medium was transferred to a black 96-well plate and 50 µL GLuc Assay solution added. Luminescence was read using a GloMax® Discover microplate reader with a 5 second integration.