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. 2009 Dec;23(12):2060–2074. doi: 10.1210/me.2009-0013

Fig. 5.

Fig. 5.

GPR54 interacts with β-arrestin-1 and -2. Densitometric analysis and representative autoradiograph showing the coimmunoprecipitation of β-arrestin-1 (A) and β-arrestin-2 (B) with wild-type FLAG-GPR54 and R331X. HEK 293 cells were transfected with 10 μg of β-arrestin-1 or -2-GFP and 10 μg of FLAG-GPR54. Cells were left untreated (gray bars) or stimulated (black bars) with 100 nm Kp-10 for 5 min at 37 C. Lysates were prepared, immunoprecipitated with mouse anti-FLAG antibody, and immunoblotted with mouse anti-GFP antibody. The expression of β-arrestin-1 or -2-GFP in 50 μg of total protein from the corresponding HEK 293 cell lysates is also shown. The data represent the mean ± se for three independent experiments. C, β-Arrestin-2-GFP overexpressed in HEK 293 cells was tested for its ability to coprecipitate with purified GST, GST-IL1, GST-IL2, GST-IL3, and GST-Ct peptides. Interaction was determined by Western blotting using a mouse anti-GFP antibody. The expression of β-arrestin-2-GFP in 50 μg of total protein from the corresponding HEK 293 cell lysates is also shown. AU, Arbitrary units. IB, Immunoblotting; IP, immunoprecipitation; WT, wild type.