Licor intercept antibody dilutent

Isolation of active RAS-GTP was performed using the Active Ras Pull-Down and Detection Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. RAS abundance was measured by Western blot. Western blot analysis of RBD pull-down lysates was performed with mouse anti-KRAS antibody (WH0003845M1, Sigma), rabbit anti-NRAS (ab167136, Abcam), rabbit anti-HRAS (ab32417, Abcam), and mouse anti-GAPDH (sc-4772, Santa Cruz Biotechnology). Input lysates were analyzed with mouse anti-pERK (675502, Biolegend) and rat anti-ERK (686902, Biolegend). All antibodies were diluted to 1/1000 in Licor intercept antibody dilutent (927–66003, Licor).

HEK293T cells were individually transfected or co-transfected with the expression plasmid for NF1-Flag, WT KRAS-GFP, or G12C KRAS-GFP. Cells were also simultaneously treated with either vehicle or 500 nM of AMG-510 for 24 h. Cells were harvested in IP Lysis/Wash Buffer (0.025 M tris-HCl, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, and 5% glycerol; pH 7.4 and 1× protease inhibitor) 24 h after transfection and treatment. Whole-cell lysates (500 μg) were precleared for 0.5 h using Control Agarose Resin slurry (Thermo Fisher Scientific). Immunoprecipitation was performed by first incubating 800 μl of HEK293T NF1-Flag precleared lysate with 200 μl of either WT KRAS-GFP, G12V KRAS-GFP or G12C KRAS-GFP precleared cell lysate. The final steps of the coimmunoprecipitation were performed using the Pierce Immunoprecipitation Kit (Thermo Fisher Scientific) with 10μg of immobilized anti-NF1 antibody (Santa Cruz Biotechnology, CA). A total of 500 μg of the cell lysate was added and incubated at room temperature under rotary agitation for 45 m. At the end of the incubation, the complexes were washed five times with lysis buffer. The western blot was probed with mouse monoclonal NF1 antibody (Santa Cruz Biotechnology) and mouse monoclonal RAS antibody (Thermo Fisher Scientific). All antibodies were diluted to 1/1000 in Licor intercept antibody dilutent (927–66003, Licor).