Figure 5. Acid wash decrease, but do not attenuate sCT mediated prolonged cAMP and β-arrestin signaling.

Effects of acid wash on cAMP production (A) and β-arrestin recruitment (B). U2OS CALCR cells were initially stimulated for 60 min with sCT or hCT using U2OS CALCR cells, then acid washed for 2×2 min and cultured for another 3, 6, 24 or 48 hours in fresh ligand free culture medium, and then assayed at end incubation. In parallel, standard assay groups of sCT and hCT were included as controls. C) Internalization rate of ¹²⁵I-sCT and ¹²⁵I-hCT stimulated CT_(a) receptors in U2OS CALCR. Cells were pre-stimulated for 45 min at 4°C with 0.25 nM ¹²⁵I-sCT or 0.25 nM ¹²⁵I-hCT for ensure receptor occupancy, then incubating for an additional 0, 15, 30, 60 and 120 min at 37°C to assess internalization. Asterisk (*) indicates significant difference between AUC sCT, Not Washed and AUC sCT, Acid Wash. Asterisk (#) indicates significant difference between AUC hCT, Not Washed and AUC hCT, Acid Wash. p<0.05 was considered to be significant. *  = p<0.05, **  = p<0.01, ***  = p<0.001. cAMP data are shown as mean ± SEM and representative of three individual conducted experiments with five replicates. β-arrestin data are shown as mean ± SEM of 3 experiments performed with five replicates. Internalization data are shown as mean ± SEM and representative of two individual conducted experiments in triplicates.