Fig. 6.

PKC activation produced an increase in βarr2 recruitment and DOR internalization in HEK293 cells. a HEK293 cells transiently expressing DOR-YFP and βarr2-Luc were pretreated with PMA (500 nM) for 60 min prior to exposure to the indicated ligands (1 μM) for 5 min and obtaining BRET readings. Results are expressed as mean ± SEM of agonist-induced changes in net BRET and correspond to five independent experiments carried out in triplicate. Data were analyzed by means of two-way ANOVA revealing an effect for PMA (p < 0.0001) and ligands (p < 0.0001). * p < 0.05; ** p < 0.001 using post hoc Bonferroni comparisons. b HEK293 cells stably expressing Flag-DORs were exposed to PMA as above and exposed to different agonists for the indicated periods of time. Internalization was expressed as % of receptors present at the membrane before exposure to agonist and represent mean ± SEM of 4–5 independent experiments carried out in triplicate. Statistical analysis by two-way ANOVA revealed an effect for PMA (p < 0.0001), for ligands (p < 0.0001) and an interaction (p < 0.0001). Post hoc Bonferroni comparisons indicated a significant effect of PMA on each agonist (p < 0.05), except for morphine