Journal: Frontiers in Cellular Neuroscience

Article Title: DYRK1A-mediated phosphorylation of GluN2A at Ser 1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

doi: 10.3389/fncel.2014.00331

Figure Lengend Snippet: Dual specificity tyrosine-phosphorylation-regulated kinase 1A mediated phosphorylation of GluN2A at S 1048 increases the surface expression of GluN2A . COS-7 cells were transiently co-transfected with plasmids to express GluN1 and wild-type (wt), or mutant versions of GFP-GluN2A (S1048A, phospho-deficient; S1048E, phospho-mimetic), in the presence or absence of HA-DYRK1A (wt, wild-type; KD, kinase-inactive). After fixing, the cells were incubated with an anti-GFP antibody to label the surface receptors (blue). Direct green fluorescence was used to measure total GFP-GluN2A expression (green). HA-DYRK1A expressing cells were identified by anti HA-immunostaining (red). Scale bar = 10 µm. The histogram represents the mean ± SEM of the GluN2A surface expression normalized to the total GFP-GluN2A signal, with the values for transfections GluN1+GFP-GluN2Awt considered as 100 ( n = 38–130 cells from, at least, three independent experiments per condition; * p < 0.05, *** p < 0.001, ANOVA).

Article Snippet: Surface expression of GFP-GluN2A was detected using an antibody against GFP (1:1000, Life Technologies Cat# A11122 RRID:AB_10073917) that recognizes the extracellular epitope of heterologously expressed receptors and that was visualized with an Alexa 647-conjugated goat anti-rabbit Ab (1:1000, Molecular Probes (Invitrogen) Cat# A21245 RRID:AB_141775).

Techniques: Expressing, Transfection, Mutagenesis, Incubation, Fluorescence, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: DYRK1A-mediated phosphorylation of GluN2A at Ser 1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

doi: 10.3389/fncel.2014.00331

Figure Lengend Snippet: Dual specificity tyrosine-phosphorylation-regulated kinase 1A mediated phosphorylation of GluN2A at S 1048 increases its surface expression in primary cortical neurons . Primary cultures of mouse embryo cortices were transiently transfected with GFP-GluN2A (wt, wild-type; S1048A, phospho-deficient mutant; S1048E, phospho-mimetic mutant) on day in vitro 8 (DIV8), in the presence or absence of heterologous HA-DYRK1A. The effect of DYRK1A on the surface:intracellular ratio of GluN1/GluN2A in primary mouse cortical neurons was evaluated by immunofluorescence. Prior to permeabilization, anti-GFP/Alexa488 was used to detect the surface chimeric receptors (represented in green), whereas intracellular GFP-GluN2A receptors were visualized after permeabilizing the cells, using an anti-GFP/Alexa555 antibody. Scale bar = 5 µm. The histogram represents the mean ± SEM GluN2A surface expression normalized to the intracellular GFP-GluN2A signal ( n = 31–52 dendrites from, at least, three independent experiments per condition, * p < 0.05, ** p < 0.01, *** p < 0.001, ANOVA).

Article Snippet: Surface expression of GFP-GluN2A was detected using an antibody against GFP (1:1000, Life Technologies Cat# A11122 RRID:AB_10073917) that recognizes the extracellular epitope of heterologously expressed receptors and that was visualized with an Alexa 647-conjugated goat anti-rabbit Ab (1:1000, Molecular Probes (Invitrogen) Cat# A21245 RRID:AB_141775).

Techniques: Expressing, Transfection, Mutagenesis, In Vitro, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: DYRK1A-mediated phosphorylation of GluN2A at Ser 1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

doi: 10.3389/fncel.2014.00331

Figure Lengend Snippet: Reduction in the internalization rate of GluN2A in the presence of DYRK1A . Live transiently co-transfected COS-7 cells were incubated with an anti-GFP antibody for 30 min at 4°C. After the rapid removal of the excess antibody, the cells were placed in the incubator for additional 30 min at 37°C to allow the GluN2A-labeled particles to be internalized. Membrane receptors were then immunolabeled with Alexa647 (shown in gray), whereas the internalized receptors were immunolabeled with Alexa555 (red particles). Scale bar = 10 µm. Bottom , Histogram representing the mean ± SEM of the normalized ratio of the internalized particles and the surface GluN2A receptors ( n = 31–115 cells from three independent experiments, *** p < 0.001, ANOVA).

Article Snippet: Surface expression of GFP-GluN2A was detected using an antibody against GFP (1:1000, Life Technologies Cat# A11122 RRID:AB_10073917) that recognizes the extracellular epitope of heterologously expressed receptors and that was visualized with an Alexa 647-conjugated goat anti-rabbit Ab (1:1000, Molecular Probes (Invitrogen) Cat# A21245 RRID:AB_141775).

Techniques: Transfection, Incubation, Labeling, Immunolabeling

Journal: Frontiers in Cellular Neuroscience

Article Title: DYRK1A-mediated phosphorylation of GluN2A at Ser 1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

doi: 10.3389/fncel.2014.00331

Figure Lengend Snippet: Dual specificity tyrosine-phosphorylation-regulated kinase 1A increases the NMDA-elicited current amplitude and opening rate. (A–D) Representative NMDA-elicited currents recorded from HEK-293T cells expressing HA-GluN1/HA-GluN2A receptors (panels A,B , wild-type GluN2A; panels C,D , GluN2A S1048A phospho-deficient mutant) in the presence (right) or absence (left) of GFP-DYRK1A. Whole-cell currents were elicited by perfusion of 1 mM NMDA with 50 µM glycine, in the continued presence of the open-channel blocker MK-801 (5 µM). (B,D) NMDA-evoked currents shown in panels (A) and (C) normalized to the same peak amplitude. (E) Average peak NMDA-evoked current density in cells transfected with NMDARs (GluN2A wt or GluN2A S1048A ) in the presence or absence of DYRK1A (* p < 0.05, ANOVA followed by Bonferroni post hoc test). (F) Average opening rate of heterologously expressed NMDARs (GluN2A wt or GluN2A S1048A ) in the presence or absence of DYRK1A (*** p < 0.001 and * p < 0.05, ANOVA followed by a Bonferroni post hoc test).

Article Snippet: Surface expression of GFP-GluN2A was detected using an antibody against GFP (1:1000, Life Technologies Cat# A11122 RRID:AB_10073917) that recognizes the extracellular epitope of heterologously expressed receptors and that was visualized with an Alexa 647-conjugated goat anti-rabbit Ab (1:1000, Molecular Probes (Invitrogen) Cat# A21245 RRID:AB_141775).

Techniques: Expressing, Mutagenesis, Transfection

Journal: Annals of neurology

Article Title: Persistent seizure control in epileptic mice transplanted with GABA progenitors

doi: 10.1002/ana.25021

Figure Lengend Snippet: Representative hippocampal sections from pilocarpine mice transplanted with MGE cells (210 DAT) labeled for DAPI and GFP (A), NeuN, GFP and GAD67 (B, top) or DAPI, GFP and Tuj1 (B, bottom). (C) Quantification of marker expression in GFP+ cells pilocarpine mice transplanted with MGE cells at 210 DAT for GAD67 and Tuj1 (n = 5 animals, each). (D) Higher resolution hippocampal images from pilocarpine mice transplanted with MGE cells (210 DAT) labeled for GFP plus parvalbumin, somatostatin or nNOS. (E) Same at 360 DAT. Arrowheads indicate double-labeled cells.

Article Snippet: Sections were double stained with a primary antibody against GFP (Aves Labs) and GAD67 (Abcam; ab97739)/TUJ (synaptic system; 302302)/SOM (Santa Cruz; sc7819)/PV (Abcam; ab11427)/CR (Millipore; AB5054)/nNOS (Millipore; AB5380)/VIP (Abcam; AB22736)/Reelin (Millipore; MAB5364).

Techniques: Labeling, Marker, Expressing