Anti c myc beads

The HEK293T cells were lysed in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA and 0.1% NP-40) supplemented with PMSF, Protease Inhibitor Cocktail and MG132, followed by rotation at 4°C for 20 min. After centrifugation, 50 μL of the supernatant was taken out as the input. Beads (Pierce™ anti c-Myc beads, Thermo scientific, 88,842; Protein A/G Magnetic beads, bimake, B23202) were washed with pre-cooled lysis buffer for three times, then the beads were incubated with supernatant at 4°C for 2–3 h. After washing with lysis buffer for 6 times, the beads were boiled in 2×SDS sample buffer for 10 min. The samples were either analyzed by Western blotting immediately as described above or stored at −80°C.

HEK 293T cells were transiently transfected in 10-cm plates with cmyc-R11.1.6, cmyc-YW1, HA-K-Ras G12D, or a combination thereof as indicated. Approximately 24 hours after transfection, cells were lysed in protease inhibitor (cOmplete EDTA-free protease inhibitor cocktail, Roche) containing NP-40 lysis buffer (Abcam). Whole cell lysates were analyzed by western blot for total HA-K-Ras G12D and B-Raf. Cmyc-tagged R11.1.6/YW1 were pulled down with anti-cmyc beads (Thermo Scientific) and analyzed for co-precipitation of HA-K-Ras G12D by western blotting and co-precipitation of other intracellular proteins by SDS-PAGE and silver stain (Thermo Scientific). Experiment was performed in duplicate. HA-tagged K-Ras G12D was pulled down with anti-HA beads (Thermo Scientific) and analyzed for co-precipitation of endogenous B-Raf by western blotting. Experiment was performed in triplicate.

Immunoprecipitations (IPs) were carried out as previously described¹⁴ . Briefly, anti-c-myc beads (Thermo Fisher Scientific) or Protein G Dynabeads (Thermo Fisher Scientific) were washed three times with phosphate-buffered saline with 0.05% Tween-20 (PBS-Tween) and resuspended in PBS-Tween containing non-immune IgG or with antibodies against the α1 subunit respectively. The antibody concentration was experimentally predetermined (Supplementary Figure 1). For α1 IPs, the antibody was crosslinked onto the beads by washing twice with 0.2 M triethanolamine (pH 8.2) (TEA), and then incubated for 30 min with 40 mM dimethyl pimelimidate (DMP) in TEA at room temperature. The beads were transferred to 50 mM Tris (pH 7.5) and incubated at room temperature for a further 15 min. The beads were washed three times with PBS-Tween and resuspended in solubilized plasma membranes in ice-cold Triton lysis buffer, supplemented with mini cOmplete protease inhibitor and PhosSTOP. The immunoprecipitation reaction was incubated overnight at 4 °C. The beads were then washed three times with PBS-Tween and eluted either with 2x sample buffer (for SDS-PAGE) or soft elution buffer [0.2% (wt/vol) SDS, 0.1% Tween-20, 50 mM Tris–HCl, pH = 8.0]¹² (for BN-PAGE).