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. 2009 Oct 15;28(23):3717–3729. doi: 10.1038/emboj.2009.300

Figure 1.

Figure 1

Identification of Tyr 1325 as one of the principal Src family kinase-mediated phosphorylation sites of the NR2A subunit. (A) Schematic diagram of GST fusion proteins containing the intracellular C-terminal region of the NR2A subunit. (B) Two-dimensional tryptic phosphopeptide maps of GST–C1, GST–C2, GST–C2–Y1325F, and GST–C3. The dot in each map shows the origin of electrophoresis. Note that P7 (Tyr 1325) was most prominently phosphorylated in vitro. (C) Identification of Tyr 1325 as one of the principal phosphorylation sites in HEK 293T cells. HEK293T cells were transfected with combinations of expression plasmids for the NR2A subunit, NR2A Y1325F mutants, and active Src (SrcYF) or inactive Src (SrcKM). NR2A immunoprecipitates from cell lysates were subjected to immunoblotting using the anti-pY antibody (4G10) (top), followed by a re-blot using the anti-NR2A antibody (middle). The expression levels of Src were confirmed by immunoblotting (bottom). *P<0.001, n=4, Student's t-test. Note that all lanes are from the same gel, but lanes originally present between lanes 2 and 3 have been omitted for brevity.