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. 2012 Mar 1;287(17):13674–13685. doi: 10.1074/jbc.M111.320705

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

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FIGURE 2. — GRIN proteins associate with Sprouty. A, GST pulldown of Sprouty2 and GRIN2. Human GRIN2 1–461 full-length and GST alone were expressed as GST fusion proteins in bacteria and purified. GST fusion proteins were bound to glutathione beads and incubated with cell lysates from HEK-293T cells exogenously expressing empty vector (pcDNA3.1 FLAG (pcDNA3.1 Flg)) or FLAG-hSprouty2 (Flg-hSprouty2). GST-hGRIN2-associated proteins (left panel) were immunoblotted with M2-FLAG antibodies, whereas HEK-293T whole cell lysates (WCL, right panel) were immunoblotted with anti-Sprouty2 antibodies. Representative immunoblots (WB) from one of three experiments are shown. B, co-immunoprecipitation (IP) of GRIN1 and GRIN2 with Sprouty2. HEK-293T lysate was immunoprecipitated with anti-HA (12C25) antibody and resolved using SDS-PAGE. Immunoprecipitates were immunoblotted with M2-FLAG (top left panel) and an anti-HA antibody (bottom left panel). Whole cell lysate was resolved using SDS-PAGE, immunoblotted with the M2-FLAG antibody (top right panel), and further blotted with the anti-HA antibody (bottom right panel). Representative immunoblots from one of three experiments are shown. Flg, FLAG. C, co-immunoprecipitation of GRIN2 with Sprouty proteins. HEK-293T cells were transfected with the indicated expression constructs, and total cell lysates were immunoprecipitated with the M2-FLAG antibody and resolved using SDS-PAGE. M2-FLAG immunoprecipitates were immunoblotted with an anti-HA antibody (panel 1) and with M2-FLAG antibody (panel 2). Whole cell lysates were immunoblotted with the anti-HA antibody and M2-FLAG antibody. Representative immunoblots from one of two experiments are shown.