glun2b n terminal  (Alomone Labs)

Journal: Journal of Neurochemistry

Article Title: Alterations of NMDAR Subunits in the Cerebrospinal Fluid Across Neurodegenerative and Immunological Disorders

doi: 10.1111/jnc.70192

Figure Lengend Snippet: N‐methyl‐D‐aspartate receptor (NMDAR) subunits GluN2A, GluN2B, GluN1, and GluN3A are present in human cerebrospinal fluid (CSF). (A) Western blot of human synaptic membranes and CSF for GluN1 (antibody AGP‐046), GluN2B (antibody AGC‐003), and GluN2A (antibody A6473). (B) Western blot of CSF [pooling CSF obtained from 3 to 4 animals, and loading same volume (12 μL)] from wild‐type mouse (WT), and a mouse KO for Grin3a (GluN3A subunit, antibody 07‐356). (C) Combination of different antibodies detecting the same subunit, either GluN1 (C‐terminal: 05‐432; N‐terminal: AGP‐046 and N308/48), GluN2B (N‐terminal: AGC‐003 and N59/36; C‐terminal: MA1‐2014), GluN2A (C‐terminal: A6473 and MA5‐27692), or GluN3A (C‐terminal: 07‐356; N‐terminal: AGC‐030). (D) Immunoprecipitation (IP) of control CSF against the four different subunits using alternative antibodies to reveal the western blot (WB). Together with the input and the bound fraction (B), a control IP (Bc) was also revealed, corresponding to the incubation of the sample performed with an irrelevant IgG of the same animal species as the specific anti‐NMDAR antibody. Arrowheads point the immunoreactive band considered for further analysis. The inputs samples were resolved in the same gels but are shown separately to optimize contrast for defining discrete bands. The uncropped blots are available as Figure .

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer for 1 h. Primary antibodies were used against GluN2B C‐terminal (mouse, 1:800 antibody dilution, Invitrogen MA1‐2014), GluN2B N‐terminal (rabbit, 1:800, Alomone AGC‐003), GluN2B N‐terminal (mouse, 1:200, NeuroMab Clone N59/36 Cat# 75‐101), GluN2A C‐terminal (rabbit, 1:800, Invitrogen A6473), GluN2A C‐terminal (rabbit, 1:800, Millipore 07‐632), GluN1 N‐terminal (guinea pig, 1:1000, Alomone AGP‐046), GluN1 C‐terminal (mouse, 1:800, Millipore 05‐432), GluN1 N‐terminal (mouse, 1:200, Hybridoma bank Clone N308/48), GluN3A Ct‐terminal (rabbit, 1:1000, Millipore 07‐356), GluN3A N‐terminal (rabbit, 1:800, Alomone AGC‐030), and Alix (mouse, 1:1000, Cell Signaling #2171).

Techniques: Western Blot, Immunoprecipitation, Control, Incubation

Journal: Journal of Neurochemistry

Article Title: Alterations of NMDAR Subunits in the Cerebrospinal Fluid Across Neurodegenerative and Immunological Disorders

doi: 10.1111/jnc.70192

Figure Lengend Snippet: Co‐immunoprecipitation of N‐methyl‐D‐aspartate receptor (NMDAR) subunits and lack of the NMDAR immunoreactivities in extracellular vesicles. (A) The specific NMDAR subunit was immunoprecipitated (IP) from human cerebrospinal fluid (CSF) with the indicated antibody and assayed in immunoblots (WB) probed with an alternative antibody against the same subunit and also with an additional antibody against a different subunit, as indicated. (B) Western blot against the four different subunits using the following antibodies: GluN1 AGP‐046, GluN2B AGC‐003, GluN2A A6473, and GluN3A 06‐356. We used CSF as input and three different fractions of the extracellular vesicles (EVs) fractionation protocol (see Section for details). Supernatant (SN) did not contain any EVs. Pellet P10K was obtained after centrifugation of 10 000 × g and contained apoptotic bodies and larger EVs. Pellet P100K was obtained after an ultracentrifugation of 100 000 × g and contained small EVs, as demonstrated by the presence of Alix, a canonical EV marker. The uncropped blot are available as Figure .

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer for 1 h. Primary antibodies were used against GluN2B C‐terminal (mouse, 1:800 antibody dilution, Invitrogen MA1‐2014), GluN2B N‐terminal (rabbit, 1:800, Alomone AGC‐003), GluN2B N‐terminal (mouse, 1:200, NeuroMab Clone N59/36 Cat# 75‐101), GluN2A C‐terminal (rabbit, 1:800, Invitrogen A6473), GluN2A C‐terminal (rabbit, 1:800, Millipore 07‐632), GluN1 N‐terminal (guinea pig, 1:1000, Alomone AGP‐046), GluN1 C‐terminal (mouse, 1:800, Millipore 05‐432), GluN1 N‐terminal (mouse, 1:200, Hybridoma bank Clone N308/48), GluN3A Ct‐terminal (rabbit, 1:1000, Millipore 07‐356), GluN3A N‐terminal (rabbit, 1:800, Alomone AGC‐030), and Alix (mouse, 1:1000, Cell Signaling #2171).

Techniques: Immunoprecipitation, Western Blot, Fractionation, Centrifugation, Marker

Journal: Journal of Neurochemistry

Article Title: Alterations of NMDAR Subunits in the Cerebrospinal Fluid Across Neurodegenerative and Immunological Disorders

doi: 10.1111/jnc.70192

Figure Lengend Snippet: N‐methyl‐D‐aspartate receptor (NMDAR) subunits GluN2A and GluN2B levels in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD). (A) Representative western blots of GluN2A (antibody A6473), GluN2B (antibody AGC‐003), and GluN1 (antibody AGP‐046) subunits in human CSF from AD ( n = 16) and non‐AD control cases ( n = 17). An internal control sample was used to normalize among different membranes. (B) Quantification of GluN2A and (C) GluN2B subunit levels were normalized to the GluN1 subunit. ROUT method identified 3 outliers from the control group when measuring GluN2B, which were removed from the analysis. Data are expressed as percentages with respect to controls. Error bars represent SEM. Exact p value obtained by t ‐test is shown. The uncropped blots are available as Figure .

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer for 1 h. Primary antibodies were used against GluN2B C‐terminal (mouse, 1:800 antibody dilution, Invitrogen MA1‐2014), GluN2B N‐terminal (rabbit, 1:800, Alomone AGC‐003), GluN2B N‐terminal (mouse, 1:200, NeuroMab Clone N59/36 Cat# 75‐101), GluN2A C‐terminal (rabbit, 1:800, Invitrogen A6473), GluN2A C‐terminal (rabbit, 1:800, Millipore 07‐632), GluN1 N‐terminal (guinea pig, 1:1000, Alomone AGP‐046), GluN1 C‐terminal (mouse, 1:800, Millipore 05‐432), GluN1 N‐terminal (mouse, 1:200, Hybridoma bank Clone N308/48), GluN3A Ct‐terminal (rabbit, 1:1000, Millipore 07‐356), GluN3A N‐terminal (rabbit, 1:800, Alomone AGC‐030), and Alix (mouse, 1:1000, Cell Signaling #2171).

Techniques: Western Blot, Control

Journal: Journal of Neurochemistry

Article Title: Alterations of NMDAR Subunits in the Cerebrospinal Fluid Across Neurodegenerative and Immunological Disorders

doi: 10.1111/jnc.70192

Figure Lengend Snippet: N‐methyl‐D‐aspartate receptor (NMDAR) subunits GluN2A, GluN2B, GluN1, and GluN3A are present in human cerebrospinal fluid (CSF). (A) Western blot of human synaptic membranes and CSF for GluN1 (antibody AGP‐046), GluN2B (antibody AGC‐003), and GluN2A (antibody A6473). (B) Western blot of CSF [pooling CSF obtained from 3 to 4 animals, and loading same volume (12 μL)] from wild‐type mouse (WT), and a mouse KO for Grin3a (GluN3A subunit, antibody 07‐356). (C) Combination of different antibodies detecting the same subunit, either GluN1 (C‐terminal: 05‐432; N‐terminal: AGP‐046 and N308/48), GluN2B (N‐terminal: AGC‐003 and N59/36; C‐terminal: MA1‐2014), GluN2A (C‐terminal: A6473 and MA5‐27692), or GluN3A (C‐terminal: 07‐356; N‐terminal: AGC‐030). (D) Immunoprecipitation (IP) of control CSF against the four different subunits using alternative antibodies to reveal the western blot (WB). Together with the input and the bound fraction (B), a control IP (Bc) was also revealed, corresponding to the incubation of the sample performed with an irrelevant IgG of the same animal species as the specific anti‐NMDAR antibody. Arrowheads point the immunoreactive band considered for further analysis. The inputs samples were resolved in the same gels but are shown separately to optimize contrast for defining discrete bands. The uncropped blots are available as Figure .

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer for 1 h. Primary antibodies were used against GluN2B C‐terminal (mouse, 1:800 antibody dilution, Invitrogen MA1‐2014), GluN2B N‐terminal (rabbit, 1:800, Alomone AGC‐003), GluN2B N‐terminal (mouse, 1:200, NeuroMab Clone N59/36 Cat# 75‐101), GluN2A C‐terminal (rabbit, 1:800, Invitrogen A6473), GluN2A C‐terminal (rabbit, 1:800, Millipore 07‐632), GluN1 N‐terminal (guinea pig, 1:1000, Alomone AGP‐046), GluN1 C‐terminal (mouse, 1:800, Millipore 05‐432), GluN1 N‐terminal (mouse, 1:200, Hybridoma bank Clone N308/48), GluN3A Ct‐terminal (rabbit, 1:1000, Millipore 07‐356), GluN3A N‐terminal (rabbit, 1:800, Alomone AGC‐030), and Alix (mouse, 1:1000, Cell Signaling #2171).

Techniques: Western Blot, Immunoprecipitation, Control, Incubation

Journal: Journal of Neurochemistry

Article Title: Alterations of NMDAR Subunits in the Cerebrospinal Fluid Across Neurodegenerative and Immunological Disorders

doi: 10.1111/jnc.70192

Figure Lengend Snippet: Co‐immunoprecipitation of N‐methyl‐D‐aspartate receptor (NMDAR) subunits and lack of the NMDAR immunoreactivities in extracellular vesicles. (A) The specific NMDAR subunit was immunoprecipitated (IP) from human cerebrospinal fluid (CSF) with the indicated antibody and assayed in immunoblots (WB) probed with an alternative antibody against the same subunit and also with an additional antibody against a different subunit, as indicated. (B) Western blot against the four different subunits using the following antibodies: GluN1 AGP‐046, GluN2B AGC‐003, GluN2A A6473, and GluN3A 06‐356. We used CSF as input and three different fractions of the extracellular vesicles (EVs) fractionation protocol (see Section for details). Supernatant (SN) did not contain any EVs. Pellet P10K was obtained after centrifugation of 10 000 × g and contained apoptotic bodies and larger EVs. Pellet P100K was obtained after an ultracentrifugation of 100 000 × g and contained small EVs, as demonstrated by the presence of Alix, a canonical EV marker. The uncropped blot are available as Figure .

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer for 1 h. Primary antibodies were used against GluN2B C‐terminal (mouse, 1:800 antibody dilution, Invitrogen MA1‐2014), GluN2B N‐terminal (rabbit, 1:800, Alomone AGC‐003), GluN2B N‐terminal (mouse, 1:200, NeuroMab Clone N59/36 Cat# 75‐101), GluN2A C‐terminal (rabbit, 1:800, Invitrogen A6473), GluN2A C‐terminal (rabbit, 1:800, Millipore 07‐632), GluN1 N‐terminal (guinea pig, 1:1000, Alomone AGP‐046), GluN1 C‐terminal (mouse, 1:800, Millipore 05‐432), GluN1 N‐terminal (mouse, 1:200, Hybridoma bank Clone N308/48), GluN3A Ct‐terminal (rabbit, 1:1000, Millipore 07‐356), GluN3A N‐terminal (rabbit, 1:800, Alomone AGC‐030), and Alix (mouse, 1:1000, Cell Signaling #2171).

Techniques: Immunoprecipitation, Western Blot, Fractionation, Centrifugation, Marker

Journal: Journal of Neurochemistry

Article Title: Alterations of NMDAR Subunits in the Cerebrospinal Fluid Across Neurodegenerative and Immunological Disorders

doi: 10.1111/jnc.70192

Figure Lengend Snippet: N‐methyl‐D‐aspartate receptor (NMDAR) subunits GluN2A and GluN2B levels in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD). (A) Representative western blots of GluN2A (antibody A6473), GluN2B (antibody AGC‐003), and GluN1 (antibody AGP‐046) subunits in human CSF from AD ( n = 16) and non‐AD control cases ( n = 17). An internal control sample was used to normalize among different membranes. (B) Quantification of GluN2A and (C) GluN2B subunit levels were normalized to the GluN1 subunit. ROUT method identified 3 outliers from the control group when measuring GluN2B, which were removed from the analysis. Data are expressed as percentages with respect to controls. Error bars represent SEM. Exact p value obtained by t ‐test is shown. The uncropped blots are available as Figure .

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer for 1 h. Primary antibodies were used against GluN2B C‐terminal (mouse, 1:800 antibody dilution, Invitrogen MA1‐2014), GluN2B N‐terminal (rabbit, 1:800, Alomone AGC‐003), GluN2B N‐terminal (mouse, 1:200, NeuroMab Clone N59/36 Cat# 75‐101), GluN2A C‐terminal (rabbit, 1:800, Invitrogen A6473), GluN2A C‐terminal (rabbit, 1:800, Millipore 07‐632), GluN1 N‐terminal (guinea pig, 1:1000, Alomone AGP‐046), GluN1 C‐terminal (mouse, 1:800, Millipore 05‐432), GluN1 N‐terminal (mouse, 1:200, Hybridoma bank Clone N308/48), GluN3A Ct‐terminal (rabbit, 1:1000, Millipore 07‐356), GluN3A N‐terminal (rabbit, 1:800, Alomone AGC‐030), and Alix (mouse, 1:1000, Cell Signaling #2171).

Techniques: 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: 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