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. 2004 Sep 23;23(20):3950–3961. doi: 10.1038/sj.emboj.7600387

Figure 4.

Figure 4

Internalisation of the V2R/V1aR heterodimer following the translocation of FRB-βarrestin2-YFP to a single FKBP-fused receptor. HEK 293T cells transfected with FRB-βarrestin2-YFP in the presence of Myc-V2R-FKBP+HA-V1aR (A) were incubated with rabbit polyclonal anti-Myc antibody A14 and mouse monoclonal anti-HA antibody 12CA5 for 1 h at 4°C. After treatment with 500 nM AP21967 for 30 min at 37°C, cells were fixed, permeabilised and labelled with Texas red-conjugated goat anti-rabbit and Alexa 633-conjugated goat anti-mouse antibodies to visualise V2R and V1aR, respectively. In (B), the internalisation extent of coexpressed HA epitope-tagged V1aR and Myc epitope-tagged receptor (V2R or δOR) was determined by measuring the cell surface receptor before and after AP21967 treatment by ELISA and expressed as the loss of cell surface expression (% of control). All values correspond to the mean±s.e.m. calculated from at least three independent experiments.