Figure 1.

Synthetic FABs that recognize β₂V₂R–βarr1 complex. A, Fab30 selectively recognizes agonist-induced β₂V₂R–βarr1 complex as assessed by co-immunoprecipitation. Sf9 cells expressing FLAG-tagged β2V2R and GRK2CAAX were stimulated with either carazolol (1 μm) or BI-167107 (100 nm), lysed, and mixed with purified βarr1 and Fab30 (or a control Fab). Subsequently, Fab was immunoprecipitated using protein L–agarose beads, and co-purification of the receptor was visualized by Western blotting (WB) using HRP-coupled anti-FLAG M2 antibody. Fabs were detected by Coomassie staining. B, densitometry-based quantification of Western blotting signal in A presented as means ± S.E. of four independent experiments normalized with respect to maximal response (treated as 100%). C, the ability of additional Fabs to recognize agonist-induced β₂V₂R–βarr1 complex assessed by co-immunoprecipitation following the protocol mentioned above. D, densitometry-based quantification of Western blotting signal in C presented as means ± S.E. of four independent experiments normalized with respect to carazolol condition (treated as 1). The data in B and D were analyzed using one-way ANOVA. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.