The physical interaction of purified β-arrestin1 with FLAG-β2ARpp WT or FLAG-β2ARpp Y219A was validated by an in vitro coimmunoprecipitation experiment. ISO (10 μM), 6 μg of FLAG receptor, and molar equivalent amounts of β-arrestin1 and Fab30 were incubated in an assay buffer containing 20 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM CaCl2, 0.1% DDM, and 0.01% CHS for 30 min at room temperature. Next, 20 μl of M1 anti-FLAG resin was added and incubated for 30 min at room temperature with rotation. Subsequently, the resin was rapidly washed three times with assay buffer and eluted with FLAG peptide (1 mg/ml), 5 mM EDTA, 20 mM Hepes (pH 7.4), and 100 mM NaCl. Coimmunoprecipitated β-arrestin1 was visualized by Coomassie staining.

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