Figure 5: Barbadin inhibits the β-arrestin- and AP2-dependent endocytosis of GPCRs.
(a–c) Cell surface expression of HA-V2R-Venus (a,c) or HA-β2AR-Venus (b) transfected in HEK293T cells was monitored by FACS following pre-incubation with DMSO, Barbadin (100 μM), Pitstop2 (100 μM) or Dyngo-4a (30 μM) for 30 min, before agonist stimulation (AVP (100 nM, a,c), ISO (10 μM, b)) at the indicated times. Data are the mean±s.e.m. of at least three independent experiments. Statistical significance of the effect of Barbadin, Pitstop 2 and Dyngo, as compared to DMSO, was assessed by a two-way ANOVA followed by Bonferroni’s post-hoc tests (**P<0.01; ***P<0.001). (d) Cell surface expression of HA-ETAR was monitored by FACS following pre-incubation with DMSO, Barbadin (100 μM) or Pitstop2 (100 μM) for 30 min, before ET1 (10 nM) stimulation for 30 min. Data are the mean±s.e.m. of at least three independent experiments. Statistical significance was assessed by a one-way ANOVA followed by Tuckey’s post-hoc tests (NS, non-significant; **P<0.01). (e,f) Native transferrin receptor (TfR) uptake was monitored by FACS in HeLa cells. Cells were pretreated with DMSO, Barbadin (100 μM), or Pitstop2 (100 μM) for 30 min, then incubated with Alexa Fluor 633-conjugated transferrin antibody (100 μg ml−1) for 30 min at 4 °C, and finally shifted to 37 °C for 15 min (e) or the indicated time (f). Data are the mean±s.e.m. of three independent experiments. Statistical significance was assessed by a one-way ANOVA followed by Tuckey’s post-hoc tests (NS, non-significant; **P<0.01; ***P<0.001).
