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. 2011 Feb;79(2):251–261. doi: 10.1124/mol.110.067363

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Fig. 4. — Coimmunoprecipitation of Tmem147 with M3Rs in cotransfected COS-7 cells. The Tmem147 construct that was used for coimmunoprecipitation studies contained an N-terminal FLAG epitope tag (Tmem147-FLAG). Likewise, the M3R construct used (human) contained three consecutive HA epitopes at its N terminus (M3R-HA). Membranes prepared from COS-7 cells that had been cotransfected with the two constructs were solubilized, and proteins were immunoprecipitated with the indicated antibodies. Immunoprecipitates were analyzed via Western blotting using an anti-FLAG (left) or an anti-HA antibody, respectively. Lanes 2, 3, 5, and 6 show the results obtained with cells coexpressing Tmem147-FLAG and M3R-HA. Lanes 1 and 4 show the results of control experiments in which membranes were prepared from COS-7 cells expressing either Tmem147-FLAG or M3R-HA and then mixed. In mock IP experiments, cell lysates were incubated with protein-A agarose in the absence of antibodies.