Fig. 6.

Co-immunoprecipitation of Venus-arrestin constructs with M₂R. HEK arrestin-2/3 KO cells [31] were co-transfected with Venus-arrestin constructs and HA-M2R-RLuc8. After 48 h, the cells were incubated in either serum-free media alone, or serum-free media with 10 μM carbamylcholine for 15 min. The cells were lysed in IP buffer (50 mM Tris-HCl pH 7.5, 2 mM EDTA, 250 mM NaCl, 10% glycerol, 0.5% NP-40, 20 mM NaF, and 1 mM NaVO₃) and the supernatant was cleared by centrifugation at max speed for 15 min, then pre-cleared with protein G agarose. The supernatant was immunoprecipitated using a rat HA antibody against HA-M2R-Rluc8. The input (IB) and immunoprecipitated material (IP) were subjected to Western blotting to detect HA and GFP, as indicated. The GFP blot of the immunoprecipitated samples was analyzed using Versadoc. The results were statistically analyzed using one-way ANOVA followed by Dunnett’s post-hoc test. The difference with KNC is shown (*, p < 0.05, n = 3). The effect of agonist treatment on Venus-arrestin-3 (K139I) was not statistically significant (n.s.).