Fig. 2

hY₂R exhibit impaired internalization properties after recycling. a Scheme of hY₂R internalization- and recycling experiment quantifying internalized peptide. b Live cell images of HEK293-HA-hY₂R-eYFP and TAMRA-NPY uptake at distinct conditions. Control cells (60 min) were treated with 100 nM peptide for 60 min at 37 °C. Stimulation with 1 μM NPY, washing and direct second stimulation (0 min RE) with 100 nM TAMRA-NPY for 60 min, second stimulation after a 60 min recycling period in the presence or absence of the recycling inhibitor NH₄Cl (60 min RE ± NH₄Cl). Ribosomal protein synthesis was inhibited by cycloheximide [CHX]. The nuclei were stained with Hoechst33342 (blue). c Quantification of TAMRA-NPY uptake. Uptake of HEK293-HA-hY₂R-eYFP cells after stimulation with 100 nM peptide for 60 min at 37 °C was set to 100% (w/o, white bar). Stimulation with 1 μM NPY, washing and direct second stimulation (0 RE, dark grey bar) or after a recycling period ± NH₄Cl (60 RE ± NH₄Cl) with 100 nM TAMRA-NPY for 60 min revealed no difference in TAMRA internalization. Scale bar: 10 μm, experiments represent data n ≥ 3; significance was determined by one-way ANOVA, Tukey post test, ns: not significant, ***: P < 0.0001