NanoBRET assay

The NanoBRET protocol was adapted from the technical manual provided in the Promega NanoBRET Protein:Protein Interaction System. For assay validation, 293FT cells were plated at a density of 4×10⁵ cells per well of a 6 well dish, and for screening purposes, cells were plated at a density of 2×10⁶ cells per 10 cm dish. The next day, cells were transfected using the Xtreme- GENE9 transfection reagent per the manufacturer’s instructions, using a 2:1 ratio of XtremeGENE9 to DNA. For the DNA constructs, an acceptor to donor ratio of 5:1 was used (125 ng pCMV-Halo-KRAS^Q61R and 25 ng pCMV-Nano-CRAF^WT per 10 cm dish or 25 ng pCMV-Halo-KRAS^Q61R and 5 ng pCMV-Nano-CRAF^WT per well of a 6 well plate). 24 hours post transfection, cells were trypsinized, washed in complete media, and re-suspended in OptiMEM containing 4% FBS at a concentration of 1.1×10⁵ cells per mL. HaloTag 618 ligand was then added to the cell suspension (1μL/mL), and 36 μL of the cell mixture was plated per well of a 384-well white-walled tissue culture plate using a μFill dispenser (BioTek), prior to incubation at 37°C. The next day, 10X stocks of each drug were prepared in phenol-free medium. 4 μL of the 10X drug stock was then added to the appropriate wells using the Biomek FX liquid handler to yield a final volume of 40 μL. For a vehicle control, DMSO was diluted in media and added to cells to make a final concentration of 0.4% DMSO. Plates were incubated with drug for 4 hours at 37°C. To measure BRET, the Nano-Glo substrate was diluted into phenol-free medium (10 μL/mL) and 10 μL of the mixture was added to each well, following which the liquid volumes were mixed for 60 seconds using an orbital shaker. Donor (460nm) and acceptor (618nm) emissions were measured within 10 minutes of substrate addition using the Envision microplate reader (Perkin Elmer) containing a 460nm/50nm emission filter and a 610nm LP filter. The BRET signal was calculated using the following formula: acceptor 618nm emission/donor 460nm emissions x 1000.

For saturation curve analysis, a 6-point saturation curve was generated in which cells plated in a 6 well dish were transfected with a constant amount (5 ng) of the energy donor construct (Nano-CRAF) and increasing amounts (0 to 100 ng) of the energy acceptor plasmid (Halo-KRAS). For competition experiments, a non-BRET-tagged construct encoding the CRAF regulatory domain (pcDNA3-FLAG-CRAF-Reg) was co-transfected into cells at increasing DNA concentrations along with the Nano-CRAF^WT (5 ng) and Halo-KRAS^Q61R (25 ng) constructs. Following transfection, cells were processed, and BRET signals determined as described above.