Antagonist binding of the β2AR-T4L-NLP complex. A) Cell-free protein expression was performed in the presence of 40 nM [3H]DHA. The unbound isotope was removed by affinity purification and binding was determined as described in methods. B) Similar reactions as in A) were performed in the presence of NLPs with or without excess of unlabeled propranolol. Two unrelated GPCR proteins 5HT1a and CHRM1 were used as negative controls. C) Similar reactions as in A) were performed in the presence of 1.5 mM [35S]Met. Proteins were separated by SDS-PAGE and visualized by autoradiography. D) Products from the reaction described in C) were separated by native electrophoresis and visualized by autoradiography. Bands with an apparent MW of 720 Kd are non-specific. Experiments in (A)-(D) were performed using the MembraneMax Protein Expression Kit (Life Technologies, Carlsbad, CA) employing DTT-free buffers with the indicated modifications (see methods).