Following 48 hours of transient overexpression of indicated flag-tagged or YFP-tagged expression constructs, we treated the cells with 1 μM or 10 μM vemurafenib, encorafenib, dabrafenib, or PLX8394 for 1, 3, or 16 hours. Subsequent to PBS washing steps, we homogenized them using a syringe with 15 strikes [standard lysis buffer: 10 mM sodium phosphate (pH 7.2), 150 mM NaCl, 0.5% Triton X-100 supplemented with standard protease inhibitors and phosphatase inhibitors]. We clarified the lysate (13,000 rpm, 20 min) and performed IPs using Protein A/G mixtures and 2 μg of control and anti–flag-tagged or GFP antibodies for 3 hours at 4°C. Resin-associated proteins were washed four times with standard lysis buffer and eluted with Laemmli sample buffer.

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