Cells were solubilized with DM detergent (Anatrace) to form micelles around membrane proteins and maintain GPCR and RAMP structure and complex formation. Twenty-four hours after transfection, transfected HEK293F cells were harvested and washed once with cold PBS. Cells were then incubated in solubilization buffer [50 mM Hepes, 1 mM EDTA, 150 mM NaCl, and 5 mM MgCl2 (pH 7.4)] with 1% (w/v) DM and cOmplete protease inhibitor (Roche) for 2 hours at 4°C with nutation. Following solubilization, lysates were clarified by centrifugation at 22,000g for 20 min at 4°C. Clarified lysates were then transferred to a microcentrifuge tube, and total protein content was determined by Protein DC assay according to the manufacturer’s specifications (Bio-Rad).

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