Figure 2.
C5apep is a partial agonist for βarr recruitment. A, HEK-293 cells expressing FLAG-C5aR1 and either βarr1 or 2 were stimulated with the indicated concentrations of different ligands (W54011, 0.1 μm; C5a, 1 μm; C5apep, 10 μm) followed by cross-linking using DSP. Subsequently, FLAG-C5aR1 was immunoprecipitated using anti-FLAG antibody agarose, and co-elution of βarrs was visualized using Western blotting. B, densitometry-based quantification of data presented in A (averages ± S.E.; n = 2) normalized with respect to signal for C5a-βarr1 condition (treated as 100%) and analyzed using two-way ANOVA. ***, p < 0.001. C, a time-course co-immunoprecipitation experiment to measure the interaction of C5aR1 with βarr2. The experiment was performed following the protocol as indicated except that cells were stimulated for different time points. D, densitometry-based quantification of the data presented in C (average ± S.E.; n = 2) normalized with respect to signal for C5a at 30 min condition (treated as 100%) and analyzed using two-way ANOVA. *, p < 0.05.