glun2b  (Alomone Labs)


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    Name:
    Anti NMDAR2B GluN2B extracellular Antibody
    Description:
    Anti NMDAR2B GluN2B extracellular Antibody AGC 003 is a highly specific antibody directed against an epitope of the rat protein The antibody can be used in western blot immunocytochemistry live cell imaging immunohistochemistry and immunoprecipitation applications It has been designed to recognize GluN2B from human rat and mouse samples
    Catalog Number:
    AGC-003
    Price:
    397.0
    Category:
    Primary Antibody
    Applications:
    Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Immunoprecipitation, Live Cell Imaging, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    25 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Alomone Labs glun2b
    Anti NMDAR2B GluN2B extracellular Antibody
    Anti NMDAR2B GluN2B extracellular Antibody AGC 003 is a highly specific antibody directed against an epitope of the rat protein The antibody can be used in western blot immunocytochemistry live cell imaging immunohistochemistry and immunoprecipitation applications It has been designed to recognize GluN2B from human rat and mouse samples
    https://www.bioz.com/result/glun2b/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glun2b - by Bioz Stars, 2021-09
    94/100 stars

    Images

    1) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00313

    Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P
    Figure Legend Snippet: Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Techniques Used: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

    Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P
    Figure Legend Snippet: Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Techniques Used: Western Blot

    2) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00313

    Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P
    Figure Legend Snippet: Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Techniques Used: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

    Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P
    Figure Legend Snippet: Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Techniques Used: Western Blot

    3) Product Images from "SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo"

    Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

    Journal: eNeuro

    doi: 10.1523/ENEURO.0175-17.2017

    CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p
    Figure Legend Snippet: CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

    Techniques Used: Infection, Western Blot

    4) Product Images from "Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells"

    Article Title: Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-87667-0

    Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p
    Figure Legend Snippet: Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p

    Techniques Used: Functional Assay, Immunofluorescence, Staining, Cell Culture

    Related Articles

    Incubation:

    Article Title: Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells
    Article Snippet: .. After 3 washes in TBS, sections were permeabilized and pre-blocked followed by primary antibody incubation at the following concentrations: platelet endothelial cell adhesion molecule (PECAM-1) (ab28364, Abcam, Cambridge, MA) (1/150), GluN1 (556308, BD Pharmingen, San Jose, CA) (1/1000) (this reference contains characterization of the antibody against GluN1), GluN2A (AGC-002, Alomone, Jerusalem, Israel) (1/20,000), GluN2B (AGC-003, Alomone, Jerusalem, Israel) (1/3000), GluN2C (ab105146, Abcam, Cambridge, MA) (1/30,000) or GluN2D (ab186816, Abcam, Cambridge, MA) (1/10,000). ..

    Article Title: Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells
    Article Snippet: .. Primary antibodies against GluN1 (Immunogen: N-terminal 42–361 amino acid) (SAB5200546, Sigma, Saint Louis, MO) and GluN2B (extracellular domain) (AGC-003, Alomone, Jerusalem, Israel) or GluN2D (N-terminal 400–500 amino acid) (ab186816, Abcam, Cambridge, MA) were added simultaneously and incubated overnight at 4 °C. ..

    Article Title: BDNF increases synaptic NMDA receptor abundance by enhancing the local translation of Pyk2 in cultured hippocampal neurons.
    Article Snippet: .. Immunocytochemistry and quantitative image analysis To label surface GluN2B-containing NMDARs, live neurons (lowdensity hippocampal cultures) were incubated for 15 min at 37°C with an antibody against an extracellular epitope of the GluN2B N terminus (1:100; AGC-003, Alomone Labs) diluted in conditioned culture medium. ..

    other:

    Article Title: Disposition of Proteins and Lipids in Synaptic Membrane Compartments Is Altered in Q175/Q7 Huntington’s Disease Mouse Striatum
    Article Snippet: AntibodiesActin (Sigma, A4700) 1:400, Alpha Actinin 2 (Abcam, ab68167) 1:2000, AMPA Receptor 1 GluR1a (Alomone, AGC-004) 1:500, BIII Tubulin (Sigma, T8660) 1:2000, Calnexin (StressGen, SPA-860) 1:2000, DARPP32 (Abcam, 40801) 1:10,000, GABA(A) a1 Receptor (Alomone, AGA-001) 1:500, Glucose Transporter GLUT3 (Abcam, ab41525) 1:500, Na+/K+ ATPase (Affinity Bioreagents, MA3-915) 1:5,000, NMDA Receptor 2B GluN2B (Alomone, AGC-003) 1:500, PDE10a (Abcam, 177933) 1:2000, PSD95 (Cell Signaling, 2507S) 1:1000, SCN4B (Abcam, ab80539) 1:200, SNAP25 (BD Transduction Labs, 610366) 1:10,000, Transferrin (Thermo Fisher, 13-6800) 1:1000, VGlut1 (Synaptic Systems, 135302) 1:10,000, VGlut2 (Synaptic Systems, 135402) 1:10,000, XK (Aviva Systems Bio, ARP33809_P050) 1:1000, Huntingtin [Ab1, aa1-17, ( )] 1:2000, poly-Q (1C2, EMD Millipore MAB1574) 1:1000.

    Immunocytochemistry:

    Article Title: BDNF increases synaptic NMDA receptor abundance by enhancing the local translation of Pyk2 in cultured hippocampal neurons.
    Article Snippet: .. Immunocytochemistry and quantitative image analysis To label surface GluN2B-containing NMDARs, live neurons (lowdensity hippocampal cultures) were incubated for 15 min at 37°C with an antibody against an extracellular epitope of the GluN2B N terminus (1:100; AGC-003, Alomone Labs) diluted in conditioned culture medium. ..

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    Alomone Labs glun2b
    Increased <t>GluN2B</t> and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P
    Glun2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glun2b/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glun2b - by Bioz Stars, 2021-09
    94/100 stars
      Buy from Supplier

    92
    Alomone Labs polyclonal rabbit anti glun2b
    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 <t>GluN2B</t> 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 Glun2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal rabbit anti glun2b/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit anti glun2b - by Bioz Stars, 2021-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    doi: 10.3389/fnmol.2018.00313

    Figure Lengend Snippet: Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Article Snippet: Surface Staining for GluN2B Living neurons were incubated for 10 min with rabbit antibodies directed against extracellular epitopes of GluN2B (diluted 1:100, Alomone Labs AGC-003), washed and fixed with 4% paraformaldehyde and 4% sucrose as described (Joshi et al., ).

    Techniques: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

    Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    doi: 10.3389/fnmol.2018.00313

    Figure Lengend Snippet: Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Article Snippet: Surface Staining for GluN2B Living neurons were incubated for 10 min with rabbit antibodies directed against extracellular epitopes of GluN2B (diluted 1:100, Alomone Labs AGC-003), washed and fixed with 4% paraformaldehyde and 4% sucrose as described (Joshi et al., ).

    Techniques: Western Blot

    Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p

    Journal: Scientific Reports

    Article Title: Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells

    doi: 10.1038/s41598-021-87667-0

    Figure Lengend Snippet: Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p

    Article Snippet: After 3 washes in TBS, sections were permeabilized and pre-blocked followed by primary antibody incubation at the following concentrations: platelet endothelial cell adhesion molecule (PECAM-1) (ab28364, Abcam, Cambridge, MA) (1/150), GluN1 (556308, BD Pharmingen, San Jose, CA) (1/1000) (this reference contains characterization of the antibody against GluN1), GluN2A (AGC-002, Alomone, Jerusalem, Israel) (1/20,000), GluN2B (AGC-003, Alomone, Jerusalem, Israel) (1/3000), GluN2C (ab105146, Abcam, Cambridge, MA) (1/30,000) or GluN2D (ab186816, Abcam, Cambridge, MA) (1/10,000).

    Techniques: Functional Assay, Immunofluorescence, Staining, Cell Culture

    Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    doi: 10.3389/fnmol.2018.00313

    Figure Lengend Snippet: Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Article Snippet: Living neurons were incubated for 10 min with rabbit antibodies directed against extracellular epitopes of GluN2B (diluted 1:100, Alomone Labs AGC-003), washed and fixed with 4% paraformaldehyde and 4% sucrose as described (Joshi et al., ).

    Techniques: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

    Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    doi: 10.3389/fnmol.2018.00313

    Figure Lengend Snippet: Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Article Snippet: Living neurons were incubated for 10 min with rabbit antibodies directed against extracellular epitopes of GluN2B (diluted 1:100, Alomone Labs AGC-003), washed and fixed with 4% paraformaldehyde and 4% sucrose as described (Joshi et al., ).

    Techniques: Western Blot

    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