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. 2008 Jan 10;27(2):361–372. doi: 10.1038/sj.emboj.7601969

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Copyright © 2008, European Molecular Biology Organization

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.

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Neuronal activity changes rapidly and bi-directionally modulate GluR2L-Thr912 and GluR4-Thr855 phosphorylation in neurons. (A) Cortical neurons were incubated in aCSF for 1 h, treated for 10 min with 1 μM okadaic acid or DMSO vehicle, and then stimulated for 10 min with 30 μM NMDA, or were left unstimulated (Con). GluR2L immunoprecipitates were blotted for phosphoThr912 (top) or GluR2L (lower panel). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) NMDA: 36.8±9.0%, N=6; NMDA+OA: 92.1±12%, N=6. GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel) or GluR4 (bottom). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to untreated control, 100%) NMDA: 28.5±6.3%, N=6; NMDA+OA: 89.3±17.4%, N=6. (B) Cortical neurons were stimulated with 20 μM Bicuculline (Bic) or 1 μM Tetrodotoxin (TTX) for 20 min. Immunoprecipitates were prepared and blotted as in (A). Lysates were blotted to detect phosphoJNK. GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) Bic: 61.9±12.8%, N=4; TTX: 142.2±16.9%, N=4. GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) Bic: 42.5±21.5%, N=4; TTX: 176.1±40.4%, N=4. (C) Cortical neurons were washed into aCSF in the presence (+) or absence (−) of 10 μM SP600125 for 1 h prior to stimulation for 5 min with 20 μM NMDA. Cells were lysed or washed into aCSF in the absence of NMDA for a further 40 min prior to lysis. Control cells were left unstimulated. GluR2L immunoprecipitates were blotted for phosphoThr912 (top), or GluR2L (second panel). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) Con/SP: 75.2±3.3%, N=4; NMDA: 58.4±7.0%, N=4; NMDA/SP: 30.1±1.8%, N=4; Rec: 85.0±12.2%, N=4; Rec/SP: 27.5± 6.6%, N=4. GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel), or GluR4 (bottom). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) Con/SP: 65.6±22.0%, N=4; NMDA: 49.3±10.8%, N=4; NMDA/SP: 30.2±9.7%, N=4; Rec: 87.4±10.7%, N=4; Rec/SP: 38.3±9.9%, N=4. Lysates were blotted for phosphoJNK (bottom). Note that the effect of SP600125 on GluR2L-Thr912 and GluR4-Thr855 phosphorylation is more marked after NMDA washout than under basal conditions (compare lanes 1,2 with 3,4 and 9,10 with 11,12).