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

Fig. 2.

Fig. 2.

GPR54 interacts with GRK2. Densitometric analysis (A) and representative autoradiograph (B) showing the coimmunoprecipitation of GRK2 with wild-type FLAG-GPR54 and R331X. HEK 293 cells were transfected with 5 μg of myc-GRK2 and 10 μg of FLAG-GPR54 or empty FLAG vector control. 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-myc antibody. The expression of myc-GRK2 in 50 μg of total protein from the corresponding HEK 293 cell lysates is also shown. The data represent the means ± se for three independent experiments. C, Purified peptides corresponding to GST-tagged GPR54 intracellular domains (GST-IL1, GST-IL2, GST-IL3, GST-Ct) were analyzed by Western blotting using a rabbit anti-GST-antibody. D, GRK2 overexpresed in HEK 293 cells was tested for its ability to coprecipitate with GST, GST-IL1, GST-IL2, GST-IL3, GST-Ct. Interaction was determined by Western blotting using a mouse anti-myc antibody. The expression of myc-GRK2 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.