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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 5. — Expression of mGRIN2 inhibits tyrosine phosphorylation of mSprouty2. A, co-immunoprecipitation of tyrosine-phosphorylated (pY) GRIN and Sprouty. Neuro-2A cells were transfected with the indicated expression constructs. Cells were serum-starved overnight. One set of cells was treated with sodium orthovanadate (1 μm) for 1 h prior to stimulation with 40 ng/ml FGF for 30 min as indicated. Total cell lysates were immunoprecipitated (IP) with M2-FLAG antibody and immunoblotted (WB) with anti-phospho-tyrosine (G410) (top panel); anti-GRIN antibody (middle panel); and M2-FLAG antibody (bottom panel). In the whole cell lysate (WCL) panels, whole cell lysate was immunoblotted with anti-GRIN antibody (panel 1) and M2-FLAG antibody (panel 2). Of note, the M2-FLAG antibody detected some nonspecific bands (panel 2), and the third band is in fact Sprouty2. mSpry2, mouse Sprouty2. Na₃VO₄, sodium orthovanadate. B, whole cell lysates from A were immunoblotted with anti-phospho-p44/42 ERK1/2 antibody (panel 1), anti-total p44/42 ERK1/2 antibody (panel 2), and anti-tubulin antibody (panel 3). Representative immunoblots from one of three independent experiments are shown.