polyclonal rabbit anti glun2a  (Alomone Labs)


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    Alomone Labs polyclonal rabbit anti glun2a
    Protein levels of mGluR1/5 and NMDAR subunits in retinal extracts from Rac1-cKO, Chat-cre +/– , and control mice. A Representative immunoblots showing the mGluR1 and mGluR5 protein levels. B Bar charts summarizing the average densitometry of immunoreactive bands of mGluR1 and mGluR5 expression. C Representative immunoblots showing the GluN1, <t>GluN2A,</t> and GluN2B protein levels. D Bar charts summarizing the average densitometry of immunoreactive bands of GluN1, GluN2A, and GluN2B expression. All the data are normalized to control. n = 6–7. * P
    Polyclonal Rabbit Anti Glun2a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal rabbit anti glun2a/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit anti glun2a - by Bioz Stars, 2022-05
    93/100 stars

    Images

    1) Product Images from "Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells"

    Article Title: Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells

    Journal: Neuroscience Bulletin

    doi: 10.1007/s12264-019-00353-0

    Protein levels of mGluR1/5 and NMDAR subunits in retinal extracts from Rac1-cKO, Chat-cre +/– , and control mice. A Representative immunoblots showing the mGluR1 and mGluR5 protein levels. B Bar charts summarizing the average densitometry of immunoreactive bands of mGluR1 and mGluR5 expression. C Representative immunoblots showing the GluN1, GluN2A, and GluN2B protein levels. D Bar charts summarizing the average densitometry of immunoreactive bands of GluN1, GluN2A, and GluN2B expression. All the data are normalized to control. n = 6–7. * P
    Figure Legend Snippet: Protein levels of mGluR1/5 and NMDAR subunits in retinal extracts from Rac1-cKO, Chat-cre +/– , and control mice. A Representative immunoblots showing the mGluR1 and mGluR5 protein levels. B Bar charts summarizing the average densitometry of immunoreactive bands of mGluR1 and mGluR5 expression. C Representative immunoblots showing the GluN1, GluN2A, and GluN2B protein levels. D Bar charts summarizing the average densitometry of immunoreactive bands of GluN1, GluN2A, and GluN2B expression. All the data are normalized to control. n = 6–7. * P

    Techniques Used: Mouse Assay, Western Blot, Expressing

    2) Product Images from "Functional Properties of Human NMDA Receptors Associated with Epilepsy-Related Mutations of GluN2A Subunit"

    Article Title: Functional Properties of Human NMDA Receptors Associated with Epilepsy-Related Mutations of GluN2A Subunit

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00155

    Relative surface expression and amplitude of whole-cell currents of NMDA receptors (NMDARs) with mutant GluN2A subunits. (A) HEK293T cells were cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and of mutant recombinant NMDARs. GRIN1 construct allowed for GluN1 subunit expression coupled with IRES-driven independent expression of eGFP fluorescent protein (green). GRIN2A constructs allowed for expression of GluN2A WT or mutant subunits fused to mcherry tag (red), which was used to estimate the total cell expression of GluN2A recombinant proteins. Surface expression of GluN2A-containing NMDARs was detected with antibodies directed to the N-terminus of GluN2A and with secondary antibodies coupled to Alexa-647 (magenta). Nuclei were counterstained with Hoescht (blue). Bar: 10 μm. Images were captured with confocal microscope. (B) Fluorescence intensities of Alexa-647 and of mCherry corresponding to the surface expression and to the total cell expression of GluN2A, respectively, were quantified within the same cells, using region of interest (ROI) manager of ImageJ software. Average relative surface expressions (RSE, ratio of surface to total expression) of WT ( n = 63 cells) and of three mutant NMDARs (p.Ile184Ser, n = 46; p.Arg518His, n = 58; p.Ala718Thr, n = 27) are represented. RSE were significantly decreased for p.Ile184Ser and p.Arg518His, but not for p.Ala718Thr, as compared with WT (Table 1 ). Kruskall Wallis test followed by Dunn’s multiple comparison test. **** p
    Figure Legend Snippet: Relative surface expression and amplitude of whole-cell currents of NMDA receptors (NMDARs) with mutant GluN2A subunits. (A) HEK293T cells were cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and of mutant recombinant NMDARs. GRIN1 construct allowed for GluN1 subunit expression coupled with IRES-driven independent expression of eGFP fluorescent protein (green). GRIN2A constructs allowed for expression of GluN2A WT or mutant subunits fused to mcherry tag (red), which was used to estimate the total cell expression of GluN2A recombinant proteins. Surface expression of GluN2A-containing NMDARs was detected with antibodies directed to the N-terminus of GluN2A and with secondary antibodies coupled to Alexa-647 (magenta). Nuclei were counterstained with Hoescht (blue). Bar: 10 μm. Images were captured with confocal microscope. (B) Fluorescence intensities of Alexa-647 and of mCherry corresponding to the surface expression and to the total cell expression of GluN2A, respectively, were quantified within the same cells, using region of interest (ROI) manager of ImageJ software. Average relative surface expressions (RSE, ratio of surface to total expression) of WT ( n = 63 cells) and of three mutant NMDARs (p.Ile184Ser, n = 46; p.Arg518His, n = 58; p.Ala718Thr, n = 27) are represented. RSE were significantly decreased for p.Ile184Ser and p.Arg518His, but not for p.Ala718Thr, as compared with WT (Table 1 ). Kruskall Wallis test followed by Dunn’s multiple comparison test. **** p

    Techniques Used: Expressing, Mutagenesis, Construct, Recombinant, Microscopy, Fluorescence, Software

    Selection of cells for whole-cell recordings. Triple transfection of human embryonic kidney (HEK) cells was performed with wild-type (WT) GRIN1-IRES-eGFP construct + WT GRIN2A-YFP construct + either of mutant GRIN2A-mCherry constructs (ratio 1:2:2). Cells with prominent fluorescence detection of all transfected subunits were selected for recordings of whole-cell currents (arrows). BF, bright field. Bar: 10 μm.
    Figure Legend Snippet: Selection of cells for whole-cell recordings. Triple transfection of human embryonic kidney (HEK) cells was performed with wild-type (WT) GRIN1-IRES-eGFP construct + WT GRIN2A-YFP construct + either of mutant GRIN2A-mCherry constructs (ratio 1:2:2). Cells with prominent fluorescence detection of all transfected subunits were selected for recordings of whole-cell currents (arrows). BF, bright field. Bar: 10 μm.

    Techniques Used: Selection, Transfection, Construct, Mutagenesis, Fluorescence

    Activation and deactivation properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 100 ms Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged activation time constants. p.Ile184Ser and p.Arg518His led to significant increases of activation time constant in both homozygous and heterozygous conditions. p.Ala716Thr had no significant effect on activation time constant in either homozygous or heterozygous conditions. Kruskal-Wallis test with Dunn’s multiple comparison test. *** p
    Figure Legend Snippet: Activation and deactivation properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 100 ms Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged activation time constants. p.Ile184Ser and p.Arg518His led to significant increases of activation time constant in both homozygous and heterozygous conditions. p.Ala716Thr had no significant effect on activation time constant in either homozygous or heterozygous conditions. Kruskal-Wallis test with Dunn’s multiple comparison test. *** p

    Techniques Used: Activation Assay, Mutagenesis, Patch Clamp, Construct, Expressing, Recombinant

    Desensitization properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 10 s Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged desensitization time constants ( τ DES ). No significant change in τ DES was detected for any condition tested. Of note, τ DES was not measurable for p.Ile184Ser in the homozygous condition, and for p.Arg518His in homozygous and heterozygous conditions because of nearly absent desensitization (Table 1 ). Kruskal-Wallis test with Dunn’s multiple comparison test. ns: not significant. (C) Averaged steady-state to peak relation ( I END10s / I P ratio). p.Ile184Ser and p.Ala716Thr had no significant effect on I END10s / I P ratio in either of homozygous or heterozygous conditions. p.Arg518His led to significant increase I END10s / I P ratio in homozygous but not in heterozygous condition. Kruskal-Wallis test with Dunn’s multiple comparison test. * p
    Figure Legend Snippet: Desensitization properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 10 s Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged desensitization time constants ( τ DES ). No significant change in τ DES was detected for any condition tested. Of note, τ DES was not measurable for p.Ile184Ser in the homozygous condition, and for p.Arg518His in homozygous and heterozygous conditions because of nearly absent desensitization (Table 1 ). Kruskal-Wallis test with Dunn’s multiple comparison test. ns: not significant. (C) Averaged steady-state to peak relation ( I END10s / I P ratio). p.Ile184Ser and p.Ala716Thr had no significant effect on I END10s / I P ratio in either of homozygous or heterozygous conditions. p.Arg518His led to significant increase I END10s / I P ratio in homozygous but not in heterozygous condition. Kruskal-Wallis test with Dunn’s multiple comparison test. * p

    Techniques Used: Mutagenesis, Patch Clamp, Construct, Expressing, Recombinant

    3) Product Images from "Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission"

    Article Title: Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69454-5

    Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p
    Figure Legend Snippet: Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

    Techniques Used: Marker

    Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p
    Figure Legend Snippet: Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p

    Techniques Used: Diffusion-based Assay

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    Alomone Labs polyclonal rabbit anti glun2a
    Protein levels of mGluR1/5 and NMDAR subunits in retinal extracts from Rac1-cKO, Chat-cre +/– , and control mice. A Representative immunoblots showing the mGluR1 and mGluR5 protein levels. B Bar charts summarizing the average densitometry of immunoreactive bands of mGluR1 and mGluR5 expression. C Representative immunoblots showing the GluN1, <t>GluN2A,</t> and GluN2B protein levels. D Bar charts summarizing the average densitometry of immunoreactive bands of GluN1, GluN2A, and GluN2B expression. All the data are normalized to control. n = 6–7. * P
    Polyclonal Rabbit Anti Glun2a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal rabbit anti glun2a/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit anti glun2a - by Bioz Stars, 2022-05
    93/100 stars
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    Protein levels of mGluR1/5 and NMDAR subunits in retinal extracts from Rac1-cKO, Chat-cre +/– , and control mice. A Representative immunoblots showing the mGluR1 and mGluR5 protein levels. B Bar charts summarizing the average densitometry of immunoreactive bands of mGluR1 and mGluR5 expression. C Representative immunoblots showing the GluN1, GluN2A, and GluN2B protein levels. D Bar charts summarizing the average densitometry of immunoreactive bands of GluN1, GluN2A, and GluN2B expression. All the data are normalized to control. n = 6–7. * P

    Journal: Neuroscience Bulletin

    Article Title: Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells

    doi: 10.1007/s12264-019-00353-0

    Figure Lengend Snippet: Protein levels of mGluR1/5 and NMDAR subunits in retinal extracts from Rac1-cKO, Chat-cre +/– , and control mice. A Representative immunoblots showing the mGluR1 and mGluR5 protein levels. B Bar charts summarizing the average densitometry of immunoreactive bands of mGluR1 and mGluR5 expression. C Representative immunoblots showing the GluN1, GluN2A, and GluN2B protein levels. D Bar charts summarizing the average densitometry of immunoreactive bands of GluN1, GluN2A, and GluN2B expression. All the data are normalized to control. n = 6–7. * P

    Article Snippet: After blocking in 5% non-fat milk at room temperature for 2 h, the membranes were incubated overnight at 4°C with the following primary antibodies: polyclonal mouse anti-GluN1 (1:1000; BD Pharmingen, Franklin Lakes, NJ), polyclonal rabbit anti-GluN2A (1:200; Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-GluN2B (1:200; Alomone Labs), polyclonal rabbit anti-GluA1 (1:200; Alomone Labs), monoclonal rabbit anti-mGluR1 (1:1000; Cell Signaling Technology, Danvers, MA), monoclonal rabbit anti-mGluR5 (1:500; Abcam), polyclonal rabbit anti-glycine receptor alpha1+alpha2 (1:1000; Abcam), and polyclonal rabbit anti-GABAA receptor alpha1 (GABRA1) (1:1000; Abcam).

    Techniques: Mouse Assay, Western Blot, Expressing

    Relative surface expression and amplitude of whole-cell currents of NMDA receptors (NMDARs) with mutant GluN2A subunits. (A) HEK293T cells were cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and of mutant recombinant NMDARs. GRIN1 construct allowed for GluN1 subunit expression coupled with IRES-driven independent expression of eGFP fluorescent protein (green). GRIN2A constructs allowed for expression of GluN2A WT or mutant subunits fused to mcherry tag (red), which was used to estimate the total cell expression of GluN2A recombinant proteins. Surface expression of GluN2A-containing NMDARs was detected with antibodies directed to the N-terminus of GluN2A and with secondary antibodies coupled to Alexa-647 (magenta). Nuclei were counterstained with Hoescht (blue). Bar: 10 μm. Images were captured with confocal microscope. (B) Fluorescence intensities of Alexa-647 and of mCherry corresponding to the surface expression and to the total cell expression of GluN2A, respectively, were quantified within the same cells, using region of interest (ROI) manager of ImageJ software. Average relative surface expressions (RSE, ratio of surface to total expression) of WT ( n = 63 cells) and of three mutant NMDARs (p.Ile184Ser, n = 46; p.Arg518His, n = 58; p.Ala718Thr, n = 27) are represented. RSE were significantly decreased for p.Ile184Ser and p.Arg518His, but not for p.Ala718Thr, as compared with WT (Table 1 ). Kruskall Wallis test followed by Dunn’s multiple comparison test. **** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Functional Properties of Human NMDA Receptors Associated with Epilepsy-Related Mutations of GluN2A Subunit

    doi: 10.3389/fncel.2017.00155

    Figure Lengend Snippet: Relative surface expression and amplitude of whole-cell currents of NMDA receptors (NMDARs) with mutant GluN2A subunits. (A) HEK293T cells were cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and of mutant recombinant NMDARs. GRIN1 construct allowed for GluN1 subunit expression coupled with IRES-driven independent expression of eGFP fluorescent protein (green). GRIN2A constructs allowed for expression of GluN2A WT or mutant subunits fused to mcherry tag (red), which was used to estimate the total cell expression of GluN2A recombinant proteins. Surface expression of GluN2A-containing NMDARs was detected with antibodies directed to the N-terminus of GluN2A and with secondary antibodies coupled to Alexa-647 (magenta). Nuclei were counterstained with Hoescht (blue). Bar: 10 μm. Images were captured with confocal microscope. (B) Fluorescence intensities of Alexa-647 and of mCherry corresponding to the surface expression and to the total cell expression of GluN2A, respectively, were quantified within the same cells, using region of interest (ROI) manager of ImageJ software. Average relative surface expressions (RSE, ratio of surface to total expression) of WT ( n = 63 cells) and of three mutant NMDARs (p.Ile184Ser, n = 46; p.Arg518His, n = 58; p.Ala718Thr, n = 27) are represented. RSE were significantly decreased for p.Ile184Ser and p.Arg518His, but not for p.Ala718Thr, as compared with WT (Table 1 ). Kruskall Wallis test followed by Dunn’s multiple comparison test. **** p

    Article Snippet: Briefly, cells were washed thrice, incubated 1 h at 4°C in PBS containing 3% bovine serum albumin (BSA), and then incubated overnight at 4°C with antibodies directed to the extracellular N-terminus of GluN2A (1:1000; Alomone) in PBS containing 3% BSA.

    Techniques: Expressing, Mutagenesis, Construct, Recombinant, Microscopy, Fluorescence, Software

    Selection of cells for whole-cell recordings. Triple transfection of human embryonic kidney (HEK) cells was performed with wild-type (WT) GRIN1-IRES-eGFP construct + WT GRIN2A-YFP construct + either of mutant GRIN2A-mCherry constructs (ratio 1:2:2). Cells with prominent fluorescence detection of all transfected subunits were selected for recordings of whole-cell currents (arrows). BF, bright field. Bar: 10 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Functional Properties of Human NMDA Receptors Associated with Epilepsy-Related Mutations of GluN2A Subunit

    doi: 10.3389/fncel.2017.00155

    Figure Lengend Snippet: Selection of cells for whole-cell recordings. Triple transfection of human embryonic kidney (HEK) cells was performed with wild-type (WT) GRIN1-IRES-eGFP construct + WT GRIN2A-YFP construct + either of mutant GRIN2A-mCherry constructs (ratio 1:2:2). Cells with prominent fluorescence detection of all transfected subunits were selected for recordings of whole-cell currents (arrows). BF, bright field. Bar: 10 μm.

    Article Snippet: Briefly, cells were washed thrice, incubated 1 h at 4°C in PBS containing 3% bovine serum albumin (BSA), and then incubated overnight at 4°C with antibodies directed to the extracellular N-terminus of GluN2A (1:1000; Alomone) in PBS containing 3% BSA.

    Techniques: Selection, Transfection, Construct, Mutagenesis, Fluorescence

    Activation and deactivation properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 100 ms Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged activation time constants. p.Ile184Ser and p.Arg518His led to significant increases of activation time constant in both homozygous and heterozygous conditions. p.Ala716Thr had no significant effect on activation time constant in either homozygous or heterozygous conditions. Kruskal-Wallis test with Dunn’s multiple comparison test. *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Functional Properties of Human NMDA Receptors Associated with Epilepsy-Related Mutations of GluN2A Subunit

    doi: 10.3389/fncel.2017.00155

    Figure Lengend Snippet: Activation and deactivation properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 100 ms Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged activation time constants. p.Ile184Ser and p.Arg518His led to significant increases of activation time constant in both homozygous and heterozygous conditions. p.Ala716Thr had no significant effect on activation time constant in either homozygous or heterozygous conditions. Kruskal-Wallis test with Dunn’s multiple comparison test. *** p

    Article Snippet: Briefly, cells were washed thrice, incubated 1 h at 4°C in PBS containing 3% bovine serum albumin (BSA), and then incubated overnight at 4°C with antibodies directed to the extracellular N-terminus of GluN2A (1:1000; Alomone) in PBS containing 3% BSA.

    Techniques: Activation Assay, Mutagenesis, Patch Clamp, Construct, Expressing, Recombinant

    Desensitization properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 10 s Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged desensitization time constants ( τ DES ). No significant change in τ DES was detected for any condition tested. Of note, τ DES was not measurable for p.Ile184Ser in the homozygous condition, and for p.Arg518His in homozygous and heterozygous conditions because of nearly absent desensitization (Table 1 ). Kruskal-Wallis test with Dunn’s multiple comparison test. ns: not significant. (C) Averaged steady-state to peak relation ( I END10s / I P ratio). p.Ile184Ser and p.Ala716Thr had no significant effect on I END10s / I P ratio in either of homozygous or heterozygous conditions. p.Arg518His led to significant increase I END10s / I P ratio in homozygous but not in heterozygous condition. Kruskal-Wallis test with Dunn’s multiple comparison test. * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Functional Properties of Human NMDA Receptors Associated with Epilepsy-Related Mutations of GluN2A Subunit

    doi: 10.3389/fncel.2017.00155

    Figure Lengend Snippet: Desensitization properties of NMDARs with mutant GluN2A subunits. Whole-cell patch clamp recordings of membrane currents were performed on HEK293T cells cotransfected with the appropriate combinations of GRIN1 and GRIN2A constructs for the expression of WT and/or of mutant recombinant NMDARs. (A) An overlay of currents evoked by 10 s Glu application in the WT (blue), homozygous (red) and heterozygous (green) conditions. Currents are normalized by peak amplitudes. (B) Averaged desensitization time constants ( τ DES ). No significant change in τ DES was detected for any condition tested. Of note, τ DES was not measurable for p.Ile184Ser in the homozygous condition, and for p.Arg518His in homozygous and heterozygous conditions because of nearly absent desensitization (Table 1 ). Kruskal-Wallis test with Dunn’s multiple comparison test. ns: not significant. (C) Averaged steady-state to peak relation ( I END10s / I P ratio). p.Ile184Ser and p.Ala716Thr had no significant effect on I END10s / I P ratio in either of homozygous or heterozygous conditions. p.Arg518His led to significant increase I END10s / I P ratio in homozygous but not in heterozygous condition. Kruskal-Wallis test with Dunn’s multiple comparison test. * p

    Article Snippet: Briefly, cells were washed thrice, incubated 1 h at 4°C in PBS containing 3% bovine serum albumin (BSA), and then incubated overnight at 4°C with antibodies directed to the extracellular N-terminus of GluN2A (1:1000; Alomone) in PBS containing 3% BSA.

    Techniques: Mutagenesis, Patch Clamp, Construct, Expressing, Recombinant

    Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

    Journal: Scientific Reports

    Article Title: Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

    doi: 10.1038/s41598-020-69454-5

    Figure Lengend Snippet: Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

    Article Snippet: Single-particle tracking For endogenous GluN2A or 2B quantum dot (QD) fluorescence particle tracking, hippocampal neurons were incubated with an antibody against the N-terminal extracellular domain of GluN2A (ACG-002 1:500, Alomone Labs) or GluN2B subunits (ACG-003 1:500, Alomone Labs) for 10 min. Neurons were then washed and incubated for 5 min with QD 655 anti-rabbit IgG (Q-11421MP, Invitrogen ).

    Techniques: Marker

    Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p

    Journal: Scientific Reports

    Article Title: Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

    doi: 10.1038/s41598-020-69454-5

    Figure Lengend Snippet: Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p

    Article Snippet: Single-particle tracking For endogenous GluN2A or 2B quantum dot (QD) fluorescence particle tracking, hippocampal neurons were incubated with an antibody against the N-terminal extracellular domain of GluN2A (ACG-002 1:500, Alomone Labs) or GluN2B subunits (ACG-003 1:500, Alomone Labs) for 10 min. Neurons were then washed and incubated for 5 min with QD 655 anti-rabbit IgG (Q-11421MP, Invitrogen ).

    Techniques: Diffusion-based Assay