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Fig. 7. Agonist-mediated endocytosis of ß₁AR and ß₂AR co-expressed in HEK 293 cells. Three different approaches were used to show the distribution of ß₁AR (left panels) and ß₂AR (middle panels) and any colocalization (right panels) in control cells (-ISO) and cells stimulated with 1 µM ISO for 15 minutes (+ISO). (A-F) Cells stably expressing HA-ß₁AR were transiently transfected with Flag-ß₂AR and arrestin-2, incubated with and without agonist, washed and fixed. The cells were then processed for immunofluorescence as described in Materials and Methods. HA-ß₁AR (red) and Flag-ß₂AR (green) are shown separately and as merged images to show colocalization (yellow). (G-L) The same as described above except the cells were incubated at 4°C with fluorescent anti-HA and anti-Flag, washed, warmed up for 5 minutes, incubated with or without agonist and fixed. HA-ß₁AR (green) and Flag-ß₂AR (red) are shown separately and as merged images to show colocalization (yellow). (M-R) Cells stably expressing HA-ß₂AR were transiently transfected with ß₁AR and arrestin-2, incubated with or without agonist, washed, fixed and processed for immunofluorescence. ß₁AR (green) and HA-ß₂AR (red) are shown separately and as merged images to show colocalization (yellow).

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