Fig. 1: Selection and characterization of Nb29 as a conformationally selective nanobody for α1AAR.
From: Structural basis of α1A-adrenergic receptor activation and recognition by an extracellular nanobody
a Flow chart of the selection process of conformationally selective nanobodies from the yeast-displayed nanobody library. For rounds 1 and 2, 0.2 μM α1AAR bound to oxymetazoline (oxy) was used for selection and alexa647-labeled anti-FLAG M1 antibody (M1-647) or biotin-labeled anti-FLAG M1 antibody Fab fragment (M1-biotin) was used for the preclear. For the FACS selection, different combinations of counterselection were performed using oxymetazoline, tamsulosin (Tam), M1-488/647, and anti-HA antibodies. b On-yeast titration to estimate the affinity of Nb29 for α1AAR, evaluated by flow cytometry. The ratio of Nb29-displayed yeast cells bound purified α1AAR in the presence or absence of 500 μM ligands was analyzed. The data represent mean ± s.e.m. of n = 3 independent measurements. c 3H-prazosin radioligand competition binding of α1AR subtype for oxymetazoline in Sf9 membranes. Samples in the presence of Nb29 were used at 5 μM concentration of Nb29. The data represent mean ± s.e.m. of n = 3 independent measurements. d, e 3H-prazosin radioligand competition binding of the purified α1AAR-bound M1-Flag affinity resin for oxymetazoline (Oxy), noradrenaline (NA) or Nb29. The data represent mean ± s.e.m. of n = 3 (NA + 5 μM Nb29), and n = 6 (the others) independent measurements. Binding affinity values are provided in Supplementary Table 1. Source data are provided in the Source Data file.
