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

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: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Schematic diagram of the mechanism underlying detection of exocytotic pH-sensitive SEP-GluN2 receptors at the neuronal surface. (B) Acidification test of SEP-GluN2A subunits expressed in DIV21 hippocampal neurons. pH values of the imaging solution are ∼7.4 and ∼5.5, respectively. Fluorescence intensity is color coded, bar=10 μm. (C) Schematic diagram showing bleaching assay in TIRF mode to detect SEP-GluN2 exocytosis. Individual exocytotic events are indicated with red arrow. (D) Representative time-lapse images of hippocampal neurons transfected with Homer1c-DsRed and SEP-GluN2A are shown to illustrate the actual detection steps of the bleaching assay for SEP-GluN2 exocytosis (step 1 and 2). Boxed regions are amplified in the upper right corner. The neurons after bleaching are outlined in yellow (step 3). An exocytosis event is annotated with red arrow in step 4. Bar=5 μm. (E) Representative maximum projection of the time-lapse image stacks showing exocytic events at 5 min. Bracketed area is amplified in upper right box. Bar=5 μm. (F) y-t projection of the region bracketed in (E) , with time-lapse frames of an exocytotic event shown in bottom panels. Scale bar as indicated. (G) Quantification of average duration of GluN2A exocytic fusion events in neurons before (control) and after 5 to 10 min of cLTP treatment (cLTP). Results are shown in mean±SEM, n.s. no significant difference, n as indicated. Neurons were from three independent preparations, two tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Imaging, Fluorescence, Bleaching Assay, Transfection, Amplification, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Schematic diagram of the cLTP model used in this study. Mature hippocampal neurons (DIV>21) were pre-incubated in Buffer A for 60 min and then flushed with 200 nM of Glycine (Buffer B) for 10 min to induce glycine-dependent cLTP. They were then returned to resting condition in Buffer A for 10 to 30 min. 50 μM APV was added to the bathing medium as indicated. Surface levels of GluN2 receptors were detected at indicated time points. (B) Calcium fluctuations of dendritic spine regions visualized in GCaMP6-expressing hippocampal neurons, which were treated with the above cLTP protocol. Fluorescence intensity of bracketed area is amplified in lower left boxes. Bar=20 μm. Kymograph (C) and intensity profile (D) of the Ca 2+ intensity in individual dendritic spines before (control) and during the 10 min of cLTP treatment (+cLTP). (E) Quantification of Ca 2+ fluctuations induced by the cLTP protocol. ⊗F/F of the GCaMP6-expressing neurons was used to show the Ca 2+ intensity change before (control), and during the cLTP treatment without (-) or with (+APV) the existence of 50 μM APV. Results are shown in scatter plot with mean±SEM, * p <0.05. n=6 (control), 7 (cLTP), 6 (+APV) cells from two independent cultures, two tailed student’s t -test. (F) Representative western blots showing the levels of biotin labeled surface GluN2A receptors in cultured rat cortical neurons treated as indicated. (G) Quantification of results in (F) . Results are shown in scatter plot with mean±SEM, * p <0.05. n=9 (ctrl), 7 (cLTP) repeats from at least seven independent preparations, two tailed student’s t -test. (H) Confocal images showing surface immunostaining for GluN2A receptors in cultured rat hippocampal neurons treated as indicated. Bar=20 μm. (I-J) Quantification of immunostaining of surface GluN2A receptor levels (I) , total GluN2A receptor levels, which is the GluN2A immunostaining signal after cell membrane permeabilization. (J) and the ratio of surface to total GluN2A receptor levels (K) before or after cLTP treatment for indicated time. Results are shown in mean±SEM, * p <0.05, ** p <0.01, *** p <0.001. Results were from n=39 (ctrl), 51 (cLTP), 42 (+APV) cells of three independent preparations, two tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Incubation, Expressing, Fluorescence, Amplification, Two Tailed Test, Western Blot, Labeling, Cell Culture, Immunostaining

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Representative confocal images of surface GluN2A immunostaining in cultured rat hippocampal neurons co-transfected with SEP-GluN2A and Homer1c-DsRed, which were treated as indicated. Boxed regions were amplified in the bottom rows. Extrasynaptic NMDARs were indicated with arrowheads. Bar=1 μm. (B) Quantification of immunostaining for surface GluN2A receptor levels. Data represent mean±SEM, n=17 (0’), 19 (-) and 22 (+APV) neurons. * p <0.05, ** p <0.01, two-tailed student’s t -test, data were derived from three independent cultures. (C) Representative 3D-SIM images of endogenous surface GluN2A and Homer immunostaining in cultured rat hippocampal neurons treated as indicated. Wide-field images are shown in the upper left insets. Boxed regions of interest (ROIs) are shown in lower panels. Extrasynaptic NMDARs were indicated with arrowheads. Bar=1 μm. (D) Cluster size of Surface GluN2A receptor were determined using the ‘surface’ function of Imaris. The frequency distribution of cluster size were shown in control, cLTP 10 min and cLTP 40 min neurons. Clusters with a diameter of 0.01 to 0.1 μm 2 were characterized as ‘small’ clusters, whereas all others were characterized as ‘big’ clusters. Data represent mean±SEM, n=6 (control), 5 (10 min) and 7 (40 min) cells were analyzed. * p <0.05, ** p <0.01, two-tailed student’s t -test, data were derived from three independent cultures. (E) Quantification of the average density of small GluN2A clusters. Data represent mean±SEM, n=12 (0’), 10 (cLTP 10’) and 14 (cLTP 40’) neurons. * p <0.05, two-tailed student’s t -test, data were derived from three independent cultures. (F) The dual-colour SIM images were analysed using Imaris software, and the representative Imaris-rendered surfaces were shown. GluN2A and Homer were extracted as separate surfaces to detect the overlap between surface of GluN2A receptors and that of the postsynaptic marker Homer. Boxed regions are amplified in lower panels. Bar=1μm. (G) Representative spatial separation efficiency of the Imaris surface method in extracting the synaptic and extrasynaptic GluN2A receptors. Non-Homer1c overlapping (Homer-) GluN2A receptors were characterized as extrasynaptic, and Homer1c overlapping (Homer+) GluN2A receptors were characterized as synaptic. Boxed regions are amplified in the bottom panels with extrasynaptic or synaptic GluN2A receptors marked using red and green surfaces, respectively. (H-I) Cumulative frequency distribution of the size of extrasynaptic (H) and synaptic (I) GluN2A receptors surfaces in cultured hippocampal neurons before (control) or after 10 min of cLTP treatment (cLTP). (J) Average cluster sizes of the synaptic and extrasynaptic surface GluN2A receptors, in cultured hippocampal neurons before (control) or after 10 min of cLTP treatment (cLTP). Data represent mean±SEM, n=606, 800, 981 and 1323 clusters from at least five neurons were compared. ** p <0.01, *** p <0.001, two-tailed student’s t -test, data were derived from three independent cultures.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Immunostaining, Cell Culture, Transfection, Amplification, Two Tailed Test, Derivative Assay, Software, Marker

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) In DIV21 rat hippocampal neurons, surface GluN2A NMDA receptors were stained with specific antibodies against their extracellular domain before fixation, followed by immunostaining using Homer-1c antibody. Confocal microscopy was used to visualize the distribution of fluorescent signals at dendritic spines. Scale bar= 5 μm. (B) The ratio of GluN2A receptors localized in the extrasynaptic region was defined as the R extra , which was calculated from the ratio of GluN2A not overlapping with Homer-1c (See Methods). Data represent mean±SEM, * p <0.05, ** p <0.01. N represents the number of cells analyzed and are shown on the bar. Data were measured from two independent cultures, two-tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Staining, Immunostaining, Confocal Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) DIV21 rat hippocampal neurons were fixed and stained with specific antibodies against endogenous GluN2A, SNAP-23 and SNAP-25. Confocal microscopy was used to detect the distribution of signals from three different fluorescent channels. To the right boxed region is amplified with channels separated. Bracketed regions are further amplified. Spine annotated by arrows were positive for GluN2A, both SNAP-23 and SNAP-25, whereas spines annotated by arrowheads shows the overlapping between GluN2A and only SNAP-23. Scale bar as indicated on the panels. (B) DIV21 rat hippocampal neurons were live-stained for surface GluN2A expression, followed by fixation and staining with specific antibodies against endogenous SNAP-23 and SNAP-25. 3D-SIM was used to visualize the distribution of fluorescent staining signals at dendritic spines. ROIs were amplified in top and right panels, with 3-color line-profile performed across the single synapse shown in right panel. Scale bar as indicated. (C) Line profiles showing the colocalization of GluN2A (green), SNAP-23 (red) and SNAP-25 (magenta). (D) Colocalization level between GluN2A and SNAP25 or SNAP23 were quantified with Pearson’s coefficient. Data represent mean ± SEM, n=43 ROIs with SNAP-25 and SNAP-23, respectively. *** p <0.001, two-tailed student’s t -test, data were measured from two independent cultures.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Staining, Confocal Microscopy, Amplification, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) PC12 cells were transfected with plasmids expressing the catalytic light-chains (Lc) of BoNT/A or BoNT/E, respectively. Cleavage of SNAP-23 and SNAP-25 was detected using their specific antibodies, full-length of SNAP-25 and SNAP-23 are indicated with arrows. (B) Representative images showing the surface level of endogenous GluN2A following 10 min cLTP stimulation in DIV21-28 rat hippocampal neurons, which were co-transfected with Homer-DsRed and BoNT/A-Lc. Boxed regions showing the level of surface GluN2A subunits were amplified in the bottom panels. Scale bar=20 μm. (C) Quantification of the surface level of endogenous GluN2A in BoNT/A-Lc transfected groups. (D) PC12 cells were transfected with shSNAP-23 or shSNAP-25 plasmids to verify knock down efficiencies. Endogenous SNAP-23 and SNAP-25 was detected using their specific antibodies. (E) Representative images showing the surface level of endogenous GluN2A following 10 min cLTP stimulation in DIV21-28 rat hippocampal neurons co-transfected with Homer-DsRed and shSNAP-23, shSNAP-25 plasmids. Boxed regions showing the level of surface GluN2A subunits were amplified in the bottom panels. Scale bar=20 μm. (F) Quantification of the surface level of endogenous GluN2A in shSNAP-23, shSNAP-25 transfected groups. Results are shown in mean±SEM, *** p <0.001, n.s. no significant difference. For E, n=47, 41, 26 and 21 cells from three independent preparations, two tailed student’s t -test. For F, n=60, 82, 25, 28, 32 and 39 cells from three independent preparations, two tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Transfection, Expressing, Amplification, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: Mature rat hippocampal neurons were transfected with BoNT/E-Lc-GFP on DIV14-17, treated with cLTP stimulation on DIV21, followed by surface GluN2A immunostaining before fixation. Representative confocal images show the typical neurodegeneration phenotype of beading axons and round cell bodies in all BoNT/E-Lc-GFP expressing neurons treated as indicated. Bar = 20 μm.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Transfection, Immunostaining, Expressing

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Representative images showing each step of the bleaching assay for the automatic analysis of exocytosis. Boxed regions are amplified in the lower left corner. Bar= 10 μm. Inset Bar = 1 μm. (B) The last step (Step 4) of the image preparation of the boxed region in (A) shows effects of standard deviation filter (see Methods). For comparison, time-lapse images of unfiltered (top panels) and SD-filtered (bottom panels) are shown. (C) Comparison of improvement in signal to noise ratio averaging of image sequence (red) or when SD-filter was applied (black). (D) The trajectories detected using TrackMate plugin of Image J. One exocytosis event is marked out with red arrow. Bar as indicated on the figures. (E) Histogram of the average duration of SEP-GluN2A exocytotic trajectories detected by automatic method. The average duration of control condition is shown as mean ± SEM, from n=50 neurons of three different preparations. R 2 =0.983 with the least squares Gaussian curve fitting method.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Bleaching Assay, Amplification, Standard Deviation, Sequencing

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Representative figures of cultured hippocampal neurons with indicated treatments showing sites of GluN2A exocytosis. Postsynaptic regions are labelled with Homer-DsRed. Boxed regions showing the exocytic events near the spine are amplified in the upper right corner. Bar=5 μm. (B) Quantification of the relative number and (C) extrasynaptic/synaptic ratio of SEP-GluN2A exocytosis in neurons before (control) and after 5 to 10 min of cLTP treatment (cLTP). (D) Quantification of exocytosis frequency in neurons before (control) and after 5-10 min of cLTP treatment (cLTP), which were compared to those of APV (50 μM)-treated or BoNT/A-Lc (A/Lc) transfected neurons. Results are shown in mean±SEM, ** p <0.01, *** p <0.001, n as indicated. Neurons were from at least three independent preparations, two tailed student’s t -test. (E) Fluorescence intensity recorded from each exocytic fusion event decayed in step-like manner. Each step corresponds to one inserted and bleached SEP-GluN2A subunit. Most intensity-time traces showed one bleaching step (left graph) and smaller number of traces had two bleaching steps (right graph). The steps are indicated with arrows. (F) A histogram showing fluorescence intensity distribution of fusion events before step bleaching has occurred. The two peaks are due to traces with one (light gray Gaussian fit) and two (dark gray Gaussian fit) bleaching steps. (G) A histogram showing the number of traces that had one, two or three bleaching steps in control (white) and cLTP treated neurons (black). Maximum number of steps observed was three. 163 (control) and 111 (cLTP) exocytic fusion events were analyzed.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Cell Culture, Amplification, Transfection, Two Tailed Test, Fluorescence