FIGURE 3.

ARRDC3 directly interacts with the β₂AR in a ligand-independent manner. Co-immunoprecipitation (A) and BRET analysis (B) were performed to assess the interaction of the β₂AR and ARRDC3 using β-arrestin2 as a control. The increased β₂AR expression level in ARRDC3 co-expressed samples shown in A, 6th and 7th lanes, is not reproducible. Cells were treated ±10 μm ISO for 25 min. Experiments were done at least three times. Representative data are shown. C, BRET analysis was performed to evaluate the interaction of the β₂AR and ARRDC3 upon treating cells with indicated ligands. BRET ratio was taken at 15 min. n = 3. Representative data are shown. Data are represented as mean ± S.D. D, bystander BRET analysis with increased amounts of GFP10-ARRDC3 to a fixed concentration of β₂AR-RlucII. n = 3. Representative data are shown. Data are represented as mean ± S.D. E, in vitro pulldown assay was performed to assess the interaction of the purified β₂AR and in vitro translated ³⁵S-labeled ARRDC3. The β₂AR was either unliganded or bound with ISO or propranolol. n = 5. Representative data are shown. Data are represented as mean ± S.E. F, representative fixed cell immunofluorescence images show the localization of the β₂AR in FLAG-β₂AR-stable HEK293 cells transiently transfected with vector or pEGFP-ARRDC3. Scale bars, 5 μm. WCL, whole cell lysate; IB, immunoblot.