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. 2014 Jul 20;13(11):3049–3062. doi: 10.1074/mcp.M114.040196

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© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Fig. 1. — Discovery of novel GLP-1R interactors. A, flow chart showing the AP-MS strategy used to identify novel GLP-1R interactors. Non-transfected (NT) CHO/MIN6 cells were used as the negative control group. CHO/MIN6 cells expressing GLP-1R-Flag were used with or without 10 nm GLP-1 stimulation for 5 min. Protein lysates from each condition were used for anti-Flag co-IP, and co-IP eluents were then used for in-solution trypsin digestion and LC-MS/MS (LTQ-XL) to profile potential GLP-1R interactors. The process was repeated three times for each condition. Among 99 potential GLP-1R interactors, 3 were selected for further validation by co-IP and western blot, immunofluorescence, and FRET. Three validated GLP-1R interactors were then selected for assessment of their effects on GIIS, leading to the discovery of one novel GLP-1R interactor of interest. B, anti-Flag immunoprecipitation and anti-Flag western blot showing that CHO cells expressed GLP-1R-Flag. C, representative immunofluorescent image showing GLP-1R-Flag expressed in CHO cells. Bar = 14 μm. D, cAMP accumulation in CHO cells expressing GLP-1R-Flag in response to GLP-1.