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. 2012 Nov 16;287(53):44234–44248. doi: 10.1074/jbc.M112.364109

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© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 9. — Co-immunoprecipitation and internalization of GPR55 and CB1R in HEK293 cells. HEK293, HEK-GPR55, HEK-CB1, and HEK-GPR55+CB1 cell lysates (A) were co-immunoprecipitated (IP) with anti-FLAG affinity matrix (IP) and immunoblotted (IB) for HA-GPR55 (1st panel). Lysates were probed for HA-GPR55 (2nd panel), FLAG-CB1 receptor (3rd panel), and β-actin (4th panel). GPR55 strongly interacts with CB1 receptors in the double expressing cell line. The blot is representative of three independent experiments. HEK-GPR55, HEK-CB1, and HEK-GPR55+CB1 cells were stimulated with 2.5 μm agonist for 45 min and receptor internalization was monitored (B). GPR55 and CB1 receptors are located on the cell surface without agonist treatment and internalize following agonist stimulation in single and double expressing cell lines. GPR55 and CB1 receptor interaction under unstimulated conditions does not alter receptor internalization. Scale bar = 20 μm.