Extended Data Fig. 3: Expression and purification of the MIPS521–ADO–A₁R–G_i2 complex.

a, Expression and purification flowchart for the A₁R–G_i2 complex. A₁R and the G_i2 heterotrimer with Gβ₁γ₂ were expressed separately in insect cell membranes. Addition of ADO (1 mM) and MIP521 (100 nM) initiated complex formation, which was solubilized with 0.5% (w/v) lauryl maltose neopentyl glycol and 0.05% (w/v) cholesteryl hemisuccinate. Solubilized A₁R and A₁R –G_i2 complex was immobilized on Flag antibody resin. Flag-eluted fractions were purified by size-exclusion chromatography (SEC). Illustrations taken from ChemDraw. b, SDS–PAGE/western blot of the purified A₁R–G_i2 complex. An anti-His antibody was used to detect Flag–A₁R-His and Gβ₁-His (red) and an anti-G_i2 antibody was used to detect Gα_i2 (green). For gel source data, see Supplementary Fig. 1. c, SDS–PAGE/Coomassie blue stain of the purified complex concentrated from the Superdex 200 Increase 10/30 column. For gel source data, see Supplementary Fig. 1. d, Representative elution profile of Flag-purified complex on Superdex 200 Increase 10/30 SEC.