Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old) incubated with BDNF (50 ng/mL). Synaptoneurosomes were immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1, PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( A ) were analyzed for the GluN2B integrated density ( B ) and GluN2B mean gray value ( C ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, in at least three independent experiments performed in different preparations. ****p < 0.0001, by Kruskal-Wallis’s test and Dunn’s multiple comparisons test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) that were stimulated with BDNF (50 ng/ml for 10 or 30 min), as indicated. Neurons were then live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A) , were analyzed for the total number ( B ), intensity ( C ), and area ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per density of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the means of the control and are the means ± SEM of 43-45 cells per condition, from at least three independent experiments performed in different preparations. *p < 0.05, **p < 0.01 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Control

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) pre-incubated with GÖ 6983 (100 nM) or vehicle (DMSO; 1:1000 dilution for 40 min) and then either maintained under the same conditions or stimulated with BDNF (50 ng/ml for 30 min), as indicated. Neurons were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A ) were analyzed for the total number ( B ), area ( C ), and intensity ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per µm of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the mean of the DMSO control and are the means ± SEM of 28 - 30 cells per condition, in at least three independent experiments performed in different preparations. ***p < 0.001, ****p < 0.0001 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation, Control

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Experimental design for the lithium-pilocarpine model of Status Epilepticus. ( B ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old, treated with saline, saline and ANA-12, Pilocarpine or Pilocarpine and ANA-12). Synaptoneurosomes were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1 and PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( B ) were analyzed for the GluN2B integrated density ( C ) and GluN2B mean gray value ( D ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, from at least four animals for each experimental condition. ****p < 0.0001, **p<0.01 as determined by Kruskal Wallis’s test and Dunn’s multiple comparisons test. Representative western blot and analysis ( E-G ). Proteins were extracted from synaptoneurosomes prepared from the same animals used in the immunocytochemistry experiments. For the immunoblot, antibodies against pTrkB, total TrKB and β-Tubulin were used. In this analysis, pTrkB levels were normalized to total TrkB levels and total TrkB levels were normalized to β-tubulin. Data are means ± SEM of at least four animals for each experimental condition. *p < 0.05, by one-way analysis of variance (ANOVA) followed by Dunnett’s post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Saline, Western Blot, Immunocytochemistry

Journal: Molecular Brain

Article Title: Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors

doi: 10.1186/s13041-015-0167-1

Figure Lengend Snippet: NMDAR internalization causes increases in serine phosphorylation of surface NMDARs. Blots shown in (a - d) are examples of experiments, in which NMDA GluN1 ( a ), GluN2A ( b and c ) or GluN2B ( d ) proteins was immunoprecipitated from the synaptic plasma membrane (LP1) of cultured neurons treated with agents in bath as indicated underneath the blots. The same filters were then stripped off and successively probed with anti-phosphoserine antibody (pSer, upper blots) and NMDAR antibodies (lower blots) for the GluN1 ( a ), GluN2A ( b and c ) or GluN2B ( d ) subunit. The ratio of band intensity showing phosphorylated versus that of total GluN1, GluN2A or GluN2B subunit proteins was normalized to the ratio in neurons treated only with culture medium (control, = 100 %, dashed line in bar graphs). Bar graphs show summary data (mean ± SEM). M: culture medium (M), V: vehicle, N: NMDA (1 mM), N + G: high NMDA/glycine (1 mM NMDA and 100 μM glycine). Effects of DHPG (50 μM) were also examined in neurons treated with DIP (50 μM, DHPG/DIP), staurosporine (1 μM, DHPG/Stau) or MPEP (10 μM, DHPG/MPEP); Effects of N + G were also examined in neurons treated with DIP (N + G/DIP), sDIP (N + G/sDIP), staurosporine (1 μM, N + G/Stau), L689560 (10 μM, N + G/L689560), monensin (10 μM, N + G/momemsin) or chloroquine (200 μM, N + G/chloroquine). #, ##: P < 0.05, 0.01 (Independent t -test) in comparison with control (M). Values in brackets indicate the number of experimental repeats

Article Snippet: The immunoprecipitates were washed 3 times with ice-cold cell lysis buffer, resuspended in 2X Laemmli sample buffer (120 mM Tris–HCl pH6.7, 4 % SDS, 10 % glycerol, 0.04 mg/ml bromophenol blue, 5 % 2-mercaptoethanol), and boiled for 5 min. Antibodies used for immunoprecipitation were: anti-GluN1 (mouse, 0.5 μg, BD Bioscience, San Jose, CA), anti-GluN2A C-terminal (rabbit, 1.5 μg, EMD Millipore, Billerica, MA), anti-GluN2A N-terminal (rabbit, 2 μg, Santa Cruz Biotech., Santa Cruz, CA), anti-GluN2B N-terminal (rabbit, 0.5 μg, Invitrogen) and anti-PKD1 (rabbit, 1 μg, Cell Signaling, Danvers, MA).

Techniques: Immunoprecipitation, Cell Culture

Journal: Molecular Brain

Article Title: Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors

doi: 10.1186/s13041-015-0167-1

Figure Lengend Snippet: Knockdown of PKD1 does not affect NMDAR internalization but prevents the NMDAR internalization-induced increases in serine phosphorylation of surface NMDARs. a - c DHPG or high NMDA/glycine (N + G) induced NMDAR internalization in neurons infected with PKD1 shRNA. After biotinylation of these neurons, GluN1 ( a ), GluN2A ( b ) or GluN2B ( c ) subunit protein was immunoprecipitated. The same filters were stripped and successively probed with HRP-conjugated streptavidin (Strep, upper blot) and an antibody against the GluN1 ( a ), GluN2A ( b ) or GluN2B ( c ) subunit (lower blots). Bar graphs show summary data (mean ± SEM) of the normalized ratios between biotinylated and total GluN1, GluN2A or GluN2B subunit proteins detected. NMDAR phosphorylation induced by bath application of DHPG or N + G in neurons infected with PKD1shRNA is shown in ( d and e ). NMDAR phosphorylation in neurons infected with control shRNA (Ctl. shRNA) is shown in ( f and g ). The same filter shown in ( d or e ) was stripped and successively probed with anti-pS1416 (top blots), anti-pSer (middle blots) and GluN2A antibody (bottom blots). The anti-pSer (upper blots) and GluN2B antibody (lower blots) were respectively used to probe the filters shown in ( e and g ). Bar graphs show summary data (mean ± SEM) of the normalized ratios between phosphorylatd and total GluN2A ( d and f ) or GluN2B ( e and g ) protein. Open and filled bars in ( d - g ) show changes detected with anti-pSer and anti-pS1416 antibodies, respectively. #, ##: P < 0.05, 0.01 (independent t -test) in comparison with control

Article Snippet: The immunoprecipitates were washed 3 times with ice-cold cell lysis buffer, resuspended in 2X Laemmli sample buffer (120 mM Tris–HCl pH6.7, 4 % SDS, 10 % glycerol, 0.04 mg/ml bromophenol blue, 5 % 2-mercaptoethanol), and boiled for 5 min. Antibodies used for immunoprecipitation were: anti-GluN1 (mouse, 0.5 μg, BD Bioscience, San Jose, CA), anti-GluN2A C-terminal (rabbit, 1.5 μg, EMD Millipore, Billerica, MA), anti-GluN2A N-terminal (rabbit, 2 μg, Santa Cruz Biotech., Santa Cruz, CA), anti-GluN2B N-terminal (rabbit, 0.5 μg, Invitrogen) and anti-PKD1 (rabbit, 1 μg, Cell Signaling, Danvers, MA).

Techniques: Infection, shRNA, Immunoprecipitation

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: Validation of the fractionation protocol in human post mortem cortex. (A) Scheme of the fractionation procedure indicating the centrifugation steps and the fractions resulting from each one. P: pellet. S: supernatant. In brief, cortical homogenates (Ho) were centrifuged a 1000× g to obtain a nuclear‐free supernatant (S1) and a pellet (P1) containing the nucleus. Centrifugation at 10,000× g of S1 resolved a supernatant that contained cell cytosol and microsomes (S2) and a pellet (P2) of plasma membranes. P2 was incubated with 1% (w/v) Triton X‐100 and centrifuged at 32,000× g to obtain a supernatant fraction collected contained extrasynaptic membranes (ExsynF); the pellet fraction was solubilized in RIPA buffer to obtain the post‐synaptic membranes (synaptic fraction, SynF). Ultracentrifugation at 100,000× g of S2 fraction served to obtain microsomal (P3) and cytosolic fractions (S3). (B). Western blot of different fractions from the fractionation protocol revealed with antibodies against synaptic‐related proteins (PSD‐95, synaptophysin), astroglial cells (glial fibrillary acidic protein [GFAP]) and no synaptic proteins associated to early endosome‐associated protein (EEA1) and to Golgi apparatus (TGN46), in control and AD samples. (C) Representative Western blot of the N‐methyl‐D‐aspartate receptor (NMDAR) subunit GluN2B, revealed with an antibody against the C‐terminal of GluN2B, of a synaptic fraction from a control sample and the quantification of the HUSPIR index for all samples (controls n = 16, Braak I–II n = 8, Braak III–IV n = 9 and Braak V–VI n = 8).

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Fractionation, Centrifugation, Incubation, Western Blot, Control

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: Characterization of N‐methyl‐D‐aspartate receptor (NMDAR) subunits in SynF and ExsynF. (A) Representative blots of the NMDAR subunits GluN2B, GluN2A, GluN1, and GluN3A from different fractions of the fractionation protocol (50 μg for S2 and extrasynaptic membranes [ExsynF]; 10 μg for P2 and synaptic fraction [SynF]). Black arrowheads indicate bands corresponding to ∼170 kDa GluN2B, ∼170 kDa GluN2A, ∼120 kDa GluN1 and ∼130 kDa GluN3A in each blot. White arrowheads indicate ∼160 kDa bands of GluN2B and GluN2A. (B) Immunoprecipitations (IP) of SynF and ExsynF of control samples. IP of GluN2B (antibody GluN2B N‐terminal, rabbit, 10 μL, Alomone AGC‐003); revealed with antibody against GluN2B C‐terminal (mouse, 1:800, Invitrogen MA1‐2014). IP of GluN2A (antibody GluN2A N‐terminal, mouse, 100 μL supernatant, HybridomaBank N327/95) revealed with antibody against GluN2A C‐terminal (rabbit, 1:800, Invitrogen A6473). IP of GluN1 (antibody GluN1 N‐terminal, guinea pig, 10 μL, Alomone AGP‐046) revealed with antibody against GluN1 N‐terminal (mouse, 30 μL supernatant, HybridomaBank, N308/48). Bc, bound from control IP (IgG); B, bound fraction from the IP; Input, SynF or ExsynF. (C) Western blot of brain homogenates from a wild‐type mouse (WT), a mouse lacking GluN3A ( Grin3a −/− ) and from control human samples (SynF and ExsynF) revealed with GluN3A ‐Ct (rabbit, 1:1000, Millipore 07‐356).

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Fractionation, Control, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: Glycosylation of N‐methyl‐D‐aspartate receptor (NMDAR) subunits. (A) Enzymatic deglycosylation of synaptic fraction (SynF) and extrasynaptic membranes (ExsynF) (3B) with N‐glycanase (N), syalidase (SA), O‐glycanase (OG), or a combination of them in control samples, revealed with antibodies against Glun2B C‐terminal (Invitrogen MA1‐2014) and GluN2A C‐terminal (Invitrogen A6473). Black arrowheads indicate bands corresponding to ∼170 kDa GluN2B and ∼170 kDa GluN2A. White arrowheads indicate ∼160 kDa bands of GluN2B and GluN2A. (B) NMDAR subunits in SynF and ExsynF fractions from control and AD cases, after N‐deglycosilation (+) or in unprocessed samples (‐), revealed with antibodies against the C‐terminal of GluN2B and GluN2A.

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Control

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: Comparison of GluN2B phosphorylation in synaptic fraction (SynF) and extrasynaptic membranes (ExsynF) between control and Alzheimer's disease (AD) cases. (A) Representative blots and (B) quantification of GluN2B (total protein resolved with mouse C‐terminal antibody MA1‐2014) and GluN2B phosphorylation (P‐GluN2B) at Tyr1472 (rabbit antibody p1516‐1472) and at Tyr1336 (rabbit antibody p1516‐1336) in synaptic and extrasynaptic GluN2B‐170 kDa from control and AD samples (Braak V–VI). The fluorescence of the secondary antibodies (IRDye 680RD goat anti‐mouse, red; IRDye 800CW goat anti‐rabbit, green) was detected with the Odyssey CLx Infrared Imaging system (LI‐COR); merge fluorescence shows co‐localization (yellow). Ratio of phosphorylated GluN2B respect to total GluN2B levels are plotted. Cases control SynF n = 9–11; control ExsynF n = 8–11; AD SynF n = 11–20; AD ExsynF n = 11–14. Observe the different Y scale for ExsynF graphs. * p < 0.05, **p < 0.001 with respect to control, t ‐test.

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Comparison, Control, Fluorescence, Imaging

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: Distribution of N‐methyl‐D‐aspartate receptor (NMDAR) subunits in membrane‐containing fractions from control and Alzheimer's disease (AD) cases. (A) Representative Western blots of NMDAR subunits in membrane fraction (P2, 10 μg), synaptic fraction (SynF, 10 μg) and extrasynaptic fractions (ExsynF, 50 μg) from control and AD samples (Braak V–VI). Tubulin was used to normalize quantifications. (B) Quantification of NMDAR subunits levels at different Braak stages and all Braak stages together (AD: Braak stages I–VI) expressed as percentage respect to controls. GluN2B‐170 kDa and GluN2A‐170 kDa levels were measured in P2, SynF and ExsynF; GluN2B‐160 kDa and GluN2A‐160 kDa were measured in ExsynF only. * p < 0.05, ** p < 0.01, *** p < 0.001 respect to control, t ‐test; # p < 0.01 analysis of variance (ANOVA) one‐way comparing control and all Braak stages. Cases control P2 n = 10–13; control SynF n = 10–14; control ExsynF n = 10–12; AD P2 n = 18–22; AD SynF n = 21–24; AD ExsynF AD n = 17–24.

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Membrane, Control, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: GluN2B phosphorylation from control and Alzheimer's disease (AD) cases comparing synaptic fraction (SynF) and extrasynaptic membranes (ExsynF). (A) Representative Western blots of GluN2B, phospho GluN2B Tyr1472, and phospho GluN2B Tyr1336 in SynF and ExsynF of controls and AD (Braak V–VI) samples. (B) Quantification of GluN2B‐170 kDa phosphorylation at SynF (phospho Tyr1472, phospho Tyr1336) and at ExsynF (phospho Tyr1336). Levels of phosphorylated GluN2B were normalized to total GluN2B and estimated as in Figure . * p < 0.05 AD v control, t ‐test. Cases control SynF n = 15–17; control ExsynF n = 13; AD SynF n = 17–22; AD ExsynF n = 19.

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Control, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: N‐Methyl‐D‐aspartate receptor (NMDAR) subunits interaction with N‐glycan lectins. (A) Representative Western blots for GluN2B, GluN2A, GluN1, and GluN3A of unbounds and inputs of synaptic fraction (SynF) and extrasynaptic membranes (ExsynF) fractions after incubation with wheat germ agglutinin (WGA) and Con A lectins, from control and Braak stage V–VI samples. (B) Quantification of SynF and ExsynF unbound fraction to WGA or Con A lectins from control and AD samples, with respect to the input fraction (SynF or ExsynF respectively) expressed as percentage (%). Data represent SynF GluN2B‐170 kDa, SynF GluN2A‐170 kDa, SynF GluN1, ExsynF GluN2B‐160 kDa, ExsynF GluN2A‐160 kDa, and ExsynF GluN1. Values represent percentage unbound ± standard deviation. Control SynF n = 5, controls ExsynF n = 7; Braak V–VI SynF n = 6, Braak V–VI ExsynF n = 7. nd , not determined.

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Western Blot, Incubation, Control, Standard Deviation

Journal: Alzheimer's & Dementia

Article Title: Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients

doi: 10.1002/alz.14125

Figure Lengend Snippet: N‐Methyl‐D‐aspartate receptor (NMDAR) subunit levels and GluN2B phosphorylation in Alzheimer's disease (AD) mouse models TauP301S and APP/PS1. (A) The fractionation protocol in wild‐type mice (WT) and transgenic mice (Tg) cortex was the same as that described for human samples in Figure . Representative Western blot of S2, P2, synaptic fraction (SynF), and extrasynaptic membranes (ExsynF) fractions from WT and TauP301S mice (Tg) developed with antibodies against Glun2B, post‐synaptic density95 (PSD95), synaptophysin, and glial fibrillary astrocytic protein (GFAP); similar patterns were obtained for APP/PS1 mice (not shown). (B) Representative Western blots of NMDAR subunits in SynF and ExsynF from WT and TauP301S mice (Tg); and from WT and APP/PS1 mice (Tg), as indicated. (C) Quantification of GluN2B, Tyr1472 phosphorylation of GluN2B (P‐GluN2B Tyr1472), Tyr1336 phosphorylation of GluN2B (P‐GluN2B Tyr1472), GluN2A, GluN1, and GluN3A levels in SynF and ExsynF from WT and TauP301S mice (Tg). WT SynF n = 6–13, WT ExsynF n = 12–13, Tg SynF n = 6–12, Tg ExsynF nn = 12. (D) Quantification of GluN2B, Tyr1472 phosphorylation of GluN2B (P‐GluN2B Tyr1472), Tyr1336 phosphorylation of GluN2B (P‐GluN2B Tyr1472), GluN2A, GluN1, and GluN3A levels in SynF and ExsynF from WT and APP/PS1 mice (Tg). WT SynF n = 5–10, WT ExsynF n = 5–10, Tg SynF n = 5–10; Tg ExsynF n = 5–10. ** p < 0.01 respect to WT.

Article Snippet: Brain extracts (100 μg in 500 μL phosphate buffered saline [PBS]) were incubated on a roller overnight at 4°C with Protein A Sepharose CL‐4B (100 μL, Cytiva 17078001) coupled with antibodies against GluN2B N‐terminal (rabbit, 15 μL, Alomone AGC‐003), GluN2B N‐terminal (mouse, 10 μL, NeuroMab 75‐097 Clone N59/20), GluN2A N‐terminal (mouse, 10 μL, Hybridoma Bank N327/95), or GluN1 N‐terminal (mouse, 10 μL, Hybridoma Bank N308/48).

Techniques: Fractionation, Transgenic Assay, Western Blot

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: The GluN2B CaMKII Site Is Dispensable for the Developmental Switch In Vitro (A) Schematic of the GluN2B ΔCaMKII amino acid changes. (B and C) Altered GluN2B phosphorylation in GluN2B ΔCaMKII/ΔCaMKII neurons. Cortical neuronal extracts were prepared and analyzed by western blot with the indicated antibodies, normalized to total GluN2B. (B) shows quantitation (mean ± SEM here and throughout), and (C) shows an example. ∗ p < 0.05 versus GluN2B +/+ (n = 4). (D) NMDAR current density at the indicated ages for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2B WT/WT : n = 14 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2B ΔCaMKII/ΔCaMKII : n = 15 [DIV7 or DIV8], n = 17 [DIV14–18]). (E) Extrasynaptic NMDAR currents were measured in GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons (n = 8 per genotype). (F and G) GluN2A and GluN2B expression analyzed by western blot at DIV8 and DIV16 for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. (F) shows quantitation and (G) shows an example. ∗ p < 0.05 versus DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (H and I) Neocortices of GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII mice were subjected to immunoprecipitation with an antibody to the N terminus of GluN2B, followed by analysis of GluN2B and CaMKIIα content. (H) shows an example and (I) shows quantitation. ∗ p < 0.05; n = 8 per genotype. (J) Percentage blockade of NMDAR currents by ifenprodil (3 μM) at the indicated stages for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2B WT/WT : n = 14 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2B ΔCaMKII/ΔCaMKII : n = 15 [DIV7 or DIV8], n = 17 [DIV14–18]). (K and L) Percentage potentiation of NMDAR currents by 100 μM spermine was measured at the indicated stages for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2B WT/WT : n = 13 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2B ΔCaMKII/ΔCaMKII : n = 18 [DIV7 or DIV8], n = 14 [DIV14–18]). (K) shows quantitation and (L) shows example traces; scale bar: 200 pA/2 s. (M and N) Neocortical extracts from P14 and P28 mice were analyzed for GluN2A expression, normalized to β-actin. (M) shows quantitation and (N) shows an example. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (O and P) Post-synaptic density (PSD) extracts from P14 and P28 mice of the indicated genotype were analyzed for GluN2A and GluN2B expression, normalized to β-actin, and the ratio calculated. (O) shows quantitation and (P) shows an example. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8).

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: In Vitro, Western Blot, Quantitation Assay, Expressing, Immunoprecipitation

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: The GluN2B CaMKII Binding Site Is Dispensable for Theta Burst LTP (A–C) Activity-dependent signaling to ERK1/2 does not require GluN2B CTD-specific sequences. DIV9 cortical neurons of the indicated genotypes were treated with TTX (500 nM), KN-62 (10 μM), or MK-801 (10 μM) for 1 hr, after which protein extracts were made and subjected to western blot analysis for phospho-ERK1/2 levels, normalized to total ERK1/2. (A) shows quantitation and (B) and (C) show example blots. ∗ p < 0.05 two-way ANOVA plus Dunnett’s post hoc test. No genotype-dependent effects were observed (p = 0.39) or genotype-drug interactions (p = 0.16; n = 8 GluN2B WT/WT ; n = 4 GluN2B ΔCaMKII/ΔCaMKII ; n = 4 GluN2B 2A(CTR)/2A(CTR) ). (D–F) Cumulative probability plots show amplitude (D) and frequency (E) of miniature EPSCs in GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII slices. Results are from 11 cells from 3 GluN2B WT/WT mice and 13 cells from 3 GluN2B ΔCaMKII/ΔCaMKII mice; p = 0.75 (amplitude); p = 0.94 (frequency); unpaired t test. (F) shows example traces. Scale bar: 10 pA/100 ms. (G) NMDAR:AMPAR EPSC ratios at −80 and +40 mV was measured in GluN2B WT/WT (n = 25; 4 mice) and GluN2B ΔCaMKII/ΔCaMKII (n = 30; 4 mice) neurons (unpaired t test; p = 0.95). (H) Weighted time constant (τw) for the decay of EPSCs recorded in (A) at +40 mV was determined using double exponential fits (unpaired t test; p = 0.63). (I) Example traces. Scale bar: 100 pA/20 ms. (J and K) Theta-burst stimulation elicited pathway-specific LTP of synaptic transmission in hippocampal CA1 area. Normalized magnitude of this potentiation 60–65 min after LTP induction did not differ significantly in GluN2B WT/WT mice (29 slices; n = 9) compared to GluN2B ΔCaMKII/ΔCaMKII (35 slices; n = 11); p = 0.162 (two-way nested ANOVA). (J) shows quantitation of data and (K) shows example traces before and after LTP induction. (K) Traces show example fEPSP traces immediately before and 1 hr after theta-burst stimulation. Scale bar: 0.5 mV/2 ms.

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Binding Assay, Activity Assay, Western Blot, Quantitation Assay, Transmission Assay

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: Distinct GluN2 CTDs Are Not Required for the 2B-to-2A Switch (A) Schematic illustrating the C-terminal domain exchange in the GluN2A 2B(CTR)/2B(CTR) mouse. (B) NMDAR current density in GluN2A WT/WT (n = 30) and GluN2A 2B(CTR)/2B(CTR) (n = 24) neurons, recorded DIV14–18. (C and D) Percentage potentiation of NMDAR currents by 100 μM spermine was measured in GluN2A WT/WT and GluN2A 2B(CTR)/2B(CTR) neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2A WT/WT : n = 19 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2A 2B(CTR)/2B(CTR) : n = 16 [DIV7 or DIV8], n = 17 [DIV14–18]). (C) shows quantitation of data and (D) shows example traces; scale bar: 200 pA/2 s. (E and F) Neocortical extracts from P14 and P28 mice of the indicated genotype were analyzed for GluN2A expression, normalized to β-actin. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (E) shows quantitation and (F) shows example blots. (G and H) PSD extracts from P14 and P28 mice of the indicated genotype were analyzed for GluN2A and GluN2B expression, normalized to β-actin, and the ratio calculated and scaled such that the ratio at P14 for WT = 1. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (G) shows quantitation and (H) shows an example blot.

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Quantitation Assay, Expressing

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: GluN2A Expression Is Sufficient to Displace GluN2B from NMDARs (A–D) Total GluN2B expression was measured in extracts from the neocortex at the indicated stages for the indicated genotypes (n = 8 per genotype). (A) and (B) show quantitation and example blots, respectively, for GluN2A WT/WT versus GluN2A 2B(CTR)/2B(CTR) . (C) and (D) show quantitation and example blots, respectively, for GluN2A WT/WT vs. GluN2B ΔCaMKII/ΔCaMKII . (E–G) Ectopic GluN2A expression is sufficient to displace GluN2B from NMDARs. Young mouse (E and F) and rat (G) neurons at DIV7 were transfected with a control (β-globin) or GluN2A-encoding vector and spermine (100 μM) potentiation of NMDAR currents measured 72 hr later. ∗ p < 0.05 (F: n = 10 of both condition; G: n = 8 control; n = 10 GluN2A). (H) Percentage potentiation of NMDAR currents by spermine (200 μM) in DIV15 or DIV16 rat cortical neurons was measured at the indicated stages for GluN2A +/+ , GluN2A +/− , and GluN2A −/− genotypes. Person r correlation coefficient: −0.9954; p = 0.031 (one-tailed test); n = 12 cells per genotype. (I) Western blot illustrating GluN2A expression in GluN2A +/+ , GluN2A +/– , and GluN2A –/– neurons. (I) A western blot confirming the absence of GluN2A expression in GluN2A −/− and an intermediate expression level in GluN2A +/− neurons.

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Expressing, Quantitation Assay, Transfection, Plasmid Preparation, One-tailed Test, Western Blot

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet:

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Recombinant