Cell signaling assays: BHK cells expressing KRASWT or KRASG12D were cultured in 10% (v/v) bovine calf

serum (Hyclone) supplemented DMEM media at 37 °C and 5% (v/v) CO2 for 24 h. The medium was
aspirated and cells were treated by compound or vehicle (DMSO) for 18 h under serum starve condition
and then analyzed by Western blotting. Cells were washed with cold phosphate-buffered saline (PBS)
and lysed in 100 μl lysis buffer (Bio-Rad) containing protease and a phosphatase inhibitor cocktail
(Roche), pH 7.4. Standard BCA protein assay was used to determine the protein concentration in each
sample. Samples were heat-denatured (95 °C) for 5 min and analyzed with Western blotting using
polyvinylidene fluoride (PVDF) membrane (Millipore). The PVDF membrane was first incubated for 1 h in
blocking solution (5% BSA in PBS with tween-20 (TBST)), and then overnight with primary antibody at 4
°C. The dilution for the primary antibody was 1:3000 for p-ERK, 1:2000 for t-ERK, 1:1000 for p-AKT, t-AKT

and p-cRaf, and 1:5000 for vinculin. Following three 5 min washes with TBST, the membrane was
incubated with a secondary antibody (1:3000) in 5% TBST for 1 h at room temperature. Signals were
detected by enhanced chemiluminescence (Thermo Fisher), imaged using X-ray film, and processed on
KODAK X-OMAT 2000 X-ray film processor. Antibodies for p-ERK, t-ERK, p-AKT, t-AKT, p-cRaf(S338) and
vinculin were from Cell Signaling Technologies Inc.

Pull-down assay: BHK cells ectopically expressing KRASG12D were cultured for 24 h in 10% (v/v) BCSS and
then lysed in lysis buffer provided in the kit. The lysates were incubated with different concentrations of
compound or DMSO for 1 h and RAS-Raf interaction was assessed with RAS binding domain (RBD) pull-
down assay using the Active Ras Pull-Down and Detection kit from Thermo Fisher.