Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-E) Postnatal development of callosal projection in S1. (A) EGFP plasmid injected into lateral ventricle of embryo at embryonic day15.5 (E15.5) and electrical pulse given to enable the plasmid to enter cortical progenitor cells of layer II/III in the ventricular zone. (B, B’) At postnatal day 5 (P5), the callosal axons from S1 had reached the white matter underneath contralateral S1. (C, C’) At P8, the callosal axons were diffusely distributed in contralateral S1. (D, D’) By P12, pruning of excess projections led to a refined innervation pattern with a narrow band limited to the S1/S2 border. (E, E’) After P12, the pattern was stable as observed at P30. (F) In P14 control mice ( Emx1 cre/+ ; NR1 fl/wt ), the callosal innervation pattern of S1 of the contralateral cortex is well-differentiated with a dense innervation at S1/S2 border. The pattern persists to P30 (J). (G) In NR1 KO mice ( Emx1 cre/+ ; NR1 fl/fl ), the innervation pattern was disrupted and projections were extremely diffuse which also persisted to P30 (K). (H) Quantification of fluorescent intensity across the medial to lateral extent of the S1. (I, L) Quantification of fluorescence density of S1 region of control vs. NR1 KO mice at P14 (I, P = 0.002) and P30 (L, P = 0.0003) Scale bar: 500μm for all images. S1: primary somatosensory cortex; S2: secondary somatosensory cortex.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Plasmid Preparation, Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: Examples of 12-μm coronal brain sections from P8 Emx1 cre/+ ; NR1 wt/wt (A) and Emx1 cre/+ ; NR1 fl/fl (B) of the same litter. Immunostaining of vesicular glutamate transporter 2 (VGult2) showed thalamocortical barrels in Layer IV of S1 which are pointed out by arrows. The VGlut2 staining in Emx1 cre/+ ; NR1 wt/wt mice revealed a clear barrel pattern (Aa). However, the barrel pattern in Emx1 cre/+ ; NR1 fl/fl mice was disrupted and less distinct (Ba). The NR1 staining in Emx1 cre/+ ; NR1 wt/wt mice were dense and strong in cortex (Ab, Ac). However, the staining in Emx1 cre/+ ; NR1 fl/fl mice was less bright and apparently reduced in Layer V and VI (Bb, Bc). Scale bar: 100μm for Ac and Bc; 500μm for rest of images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, B) The callosal axons in S1 formed a bundle and grew into the ipsilateral CC at P0 in control and NR1 KO littermates ( Emx1 cre/+ ; NR1 fl/wt and Emx1 cre/+ ; NR1 fl/fl mice). The arrows show the extent of axon growth into the CC. By P3, the callosal axons crossed the midline (C, D) and by P5, the callosal axons have grown to underneath the contralateral S1 (E, F). Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, A’) At P6, most axons in control grew into deeper layer VI of S1 (see “*”); a few axons grew to layer V from medial to lateral S1 (see arrows). However, axons projecting to lateral S2 had grown to layer IV which was apparently faster than the axons in S1 (see arrows). (B, B’) In NR1 KO mice, most axons had grown to layer V and some even grew to layer I (see arrows) at P6. (C, D) At P8, axons in control and mutant mice had grown to the superficial layer of cortex. However, the innervation patterns were different. Controls showed more axon innervation in the lateral S1 with dense callosal innervation at S1/S2 border (C). Mutants showed slightly more axon innervation in the medial S1 (D). (E) The fluorescence density of mutant mice in S1 was significantly higher than in control mice at P6 which suggested that the mutants had increased axon innervation in contralateral S1 at P6. P = 0.003. Scale bar: 500μm for all images. The square brackets in all images outline the S1. The arrow heads in all images outline the S1/S2 border. White lines outline different layers in the cortex of Figure A-D. M: medial; L: lateral.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) In control mice ( Emx1 cre/+ ; NR1 fl/wt ), cleaved caspase-3 + cells were mostly detected in layer II/III of M1 (A’), only rare cell death was observed in other cortical regions, such as S1 (A’’). (B) Compared with controls, there was increased cell death in layer II/III of motor cortex in mutant mice ( Emx1 cre/+ ; NR1 fl/fl ) (B’). However, compared with controls, there was no increased cell death in other cortical regions in mutant mice, such as S1 (B’’). Scale bar: 500μm for A and B; 200μm for A’, A’’, B’ and B’’.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Deleting NMDAR specifically in projecting neurons. Vectors expressing Cre-recombinase (Cre) and EGFP were delivered into S1 of floxed NR1 mice ( NR1 fl/wt x NR1 fl/wt ) by in utero electroporation at E15.5 (A). Callosal innervation patterns at P14 in control NR1 ipsiS1+/+ mice (B) and NR1 ipsiS1−/− mice (C). (D) Quantification of fluorescence density. P = 0.317. (E-H) Deleting NMDAR specifically in target neurons. NR1 was deleted in target contralateral S1 by in utero electroporation of Cre at E12.5 in NR1 fl/fl ; Ai14 fl/fl mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5 (E). Compared with control NR1 wt/wt ; Ai14 fl/fl (F), NR1 fl/fl ; Ai14 fl/fl mice which specifically deleted NR1 in target S1 showed increased callosal innervation in S1 as “*” shows (G). (H) Quantification of fluorescence density. P = 0.002. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Expressing, In Utero, Electroporation, Control, Fluorescence, Labeling

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) NR1 was deleted in target contralateral S1 by in utero electroporation of Cre at E13.5 in NR1 fl/fl ; Ai14 fl/fl mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5. Compared with control NR1 wt/wt ; Ai14 fl/fl (B), NR1 fl/fl ; Ai14 fl/fl , with NR1 specifically deleted in upper cortical layers did not show increased callosal innervation in (C). (D) Quantification of fluorescence density. P = 0.27. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: In Utero, Electroporation, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P8 and mice were perfused 3 hours later after last injection. Rabbit IgG served as control. Mouse brains then were stained with anti-Rabbit secondary coupled to Alexa594. The red fluorophore of Alexa594 indicated where the antibodies had distributed to. Scale bar: 500μm for all images. (B, B’) In control, the fluorescence signals were mostly detected in the cortex of the ipsilateral injection side, and few in the contralateral cortex. In the ipsilateral injection side, the signals were detected in all the cortical layers, but most strongly in the pia, layer I, layer V, layer VI, cingulum and corpus callosum (see arrows). The signals were also detected in the hippocampus and contralateral motor cortex (see arrows). (C, C’) The general antibody distribution pattern was similar as seen in control. Moreover, the anti-NR1 antibody can bind to NMDAR on the cell membranes, which thus showing beautiful cell membrane staining (see arrows in C’). Scale bar: 500 μm for Figure B, C; 200 μm for Figure B’, C’. CC: corpus callosum; cg: cingulum; Hip: hippocampus; M: motor cortex; S1: primary somatosensory cortex; S2: secondary somatosensory cortex.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Staining, Fluorescence, Membrane

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P12 in ipsilateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice did not show increased callosal innervation in S1 at P14 (C). (D) Quantification of fluorescence density. P = 0.94. (E-H) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P12 in contralateral cortex. Compared with control (F), antibody injection in mice showed increased callosal innervation in S1 at P14 (see “*”, G). (H) Quantification of fluorescence density. P =0.0002. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Anti-NR1 antibodies were injected into the lateral ventricle from P4 to P8 in contralateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice show increased callosal innervation in S1 at P14 (C). (D) Quantification of fluorescence density. P = 0.004. (E-H) Anti-NR1 antibodies were injected into the lateral ventricle from P8 to P14 in contralateral cortex. Compared with control (F), antibody injection in mice did not show increased callosal innervation in S1 at P14 (G). (H) Quantification of fluorescence density. P = 0.69. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) The callosal innervation pattern in S1 at P30 in control mice ( Emx1 cre/+ ; NR2A fl/wt ) is similar as the pattern in P14 WT control mice, with few axons in S1 but a dense innervation at S1/S2 border. (B) In the mutant mice ( Emx1 cre/+ ; NR2A fl/fl ), the general innervation pattern was as same as control. However, the increased callosal innervation at the border of M1 and S1 was persistent at P30 (see “*” in B’). (C) Quantification of fluorescence density. P = 0.63. (D) In control Emx1 cre/+ ; NR2B fl/wt mice, the callosal innervation pattern at P30 was as normal as WT control. (E) However, the increased callosal innervation in Emx1 cre/+ ; NR2B fl/fl mice lasted at least to P30 as we observed in Emx1 cre/+ ; NR1 fl/fl mice at P30. (F) Quantification of fluorescence density. P = 0.007. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, B) EPHB2 protein expression are decreased in Emx1 cre/+ ; NR1 fl/fl mice at P5. In control Emx1 cre/+ ; NR1 wt/wt mice, EPHB2 was expressed both in CC and cortex (A). EPHB2 in Emx1 cre/+ ; NR1 fl/fl mice was dramatically decreased in cortex (B). (C) Western blot analysis of cortical protein extracts from P8 S1 showed that, relative to the loading control beta-tubulin (β-Tub) and GAPDH, lower levels of EPHB2 were observed in the five samples of Emx1 cre/+ ; NR1 fl/fl mice compared to the five samples of controls. (D) Quantification of protein levels relative to β-Tub. P=0.001. (E) Quantification of protein levels relative to GAPDH. P< 0.0001. (F) The quantitative polymerase chain reaction (qPCR) analysis showed no expression difference of EPHB2 between Emx1 cre/+ ; NR1 fl/fl mice and controls. Scale bar: 500μm for A, B.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Brain Pathology

Article Title: Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

doi: 10.1111/bpa.13005

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: GluN1 , Recombinant protein corresponding to AA 660 to 811 from rat GluN1 , Synaptic Systems, Mouse, 114‐011, RRID:AB_887750 , 1:500 , 1:200 , 1:100.

Techniques: Recombinant, Purification

Journal: Brain Pathology

Article Title: Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

doi: 10.1111/bpa.13005

Figure Lengend Snippet: Western blot against human hippocampus homogenates probed with glutamatergic receptor subunit GluA2, GluN1 and GluN2A antibodies (A). The use of the GluN2A blocking peptide (BLP‐GC002) (B) and omission of the primary antibodies (C) resulted in a complete absence of immunoreactivity except for a small amount of background lipofuscin staining. Section were stained with goat anti‐rabbit Alexa Fluor 647 (B,b; C,b) and goat anti‐mouse Alexa Fluor 488 (C,c). Nuclei were counterstained with Hoechst dye (blue) (B,a; C,a). Scale bars B–C = 50 µm

Article Snippet: GluN1 , Recombinant protein corresponding to AA 660 to 811 from rat GluN1 , Synaptic Systems, Mouse, 114‐011, RRID:AB_887750 , 1:500 , 1:200 , 1:100.

Techniques: Western Blot, Blocking Assay, Staining

Journal: Brain Pathology

Article Title: Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

doi: 10.1111/bpa.13005

Figure Lengend Snippet: Summary of results for fluorescent immunohistochemistry in the human AD hippocampus subiculum, entorhinal cortex and STG

Article Snippet: GluN1 , Recombinant protein corresponding to AA 660 to 811 from rat GluN1 , Synaptic Systems, Mouse, 114‐011, RRID:AB_887750 , 1:500 , 1:200 , 1:100.

Techniques: Immunohistochemistry

Journal: Brain Pathology

Article Title: Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

doi: 10.1111/bpa.13005

Figure Lengend Snippet: GluN1 expression in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus in human control and Alzheimer's disease cases visualized by 3,3′‐diaminobenzidine‐peroxidase immunohistochemistry. Staining appeared moderate within the str. pyramidale of the CA regions (A–C), with an increase in expression within these regions and the stratum (str.) moleculare of the dentate gyrus in AD (E1,2). Arrows indicating localization to processes and around pyramidal neurons. CA, cornu ammonis; DG, dentate gyrus; ECx, entorhinal cortex; HP, hippocampus; STG, superior temporal gyrus; str. pyr, stratum pyramidale; str. rad, stratum radiatum; str. gran, stratum granulosum; Sub, subiculum. Scale bars: A1,2 = 1000 µm; B1–E1, B2–E2 = 100 µm; F1–H1, F2–H2 = 400 µm

Article Snippet: GluN1 , Recombinant protein corresponding to AA 660 to 811 from rat GluN1 , Synaptic Systems, Mouse, 114‐011, RRID:AB_887750 , 1:500 , 1:200 , 1:100.

Techniques: Expressing, Control, Immunohistochemistry, Staining

Journal: Brain Pathology

Article Title: Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

doi: 10.1111/bpa.13005

Figure Lengend Snippet: GluN1 expression is altered in hippocampal subfields in Alzheimer's disease. Photomicrographs of representative regions of the CA1(A), CA2 (B), CA3 (C), and dentate gyrus (D) showing GluN1 (red) and GluN1overlaid with NeuN (green) immunoreactivity for representative Alzheimer's disease and control cases. AD, Alzheimer's disease; CA, cornu ammonis; DG, dentate gyrus; str. ori, straum oriens; str. pyr, stratum pyramidale; str. rad, stratum radiatum; str. mol, stratum moleculare; str. gran, stratum granulosum. Scale bars A–C = 100 µm, D = 50 µm

Article Snippet: GluN1 , Recombinant protein corresponding to AA 660 to 811 from rat GluN1 , Synaptic Systems, Mouse, 114‐011, RRID:AB_887750 , 1:500 , 1:200 , 1:100.

Techniques: Expressing, Control

Journal: Brain Pathology

Article Title: Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

doi: 10.1111/bpa.13005

Figure Lengend Snippet: GluN1 expression in the subiculum, entorhinal cortex and superior temporal gyrus in human control and Alzheimer's disease cases. Photomicrographs of representative regions of the subiculum (A), entorhinal cortex (B), and superior temporal gyrus (C) showing GluN1 (red) and GluN1overlaid with NeuN (green) immunoreactivity for representative Alzheimer's disease and control cases. AD, Alzheimer's disease; CA, cornu ammonis; DG, dentate gyrus; ECx, entorhinal cortex: STG, superior temporal gyrusstr; ori, straum oriens; str. pyr, stratum pyramidale; str. rad, stratum radiatum; str. mol, stratum moleculare; str. gran, stratum granulosum. Scale bars A–C, E–G = 100 µm, D = 50 µm

Article Snippet: GluN1 , Recombinant protein corresponding to AA 660 to 811 from rat GluN1 , Synaptic Systems, Mouse, 114‐011, RRID:AB_887750 , 1:500 , 1:200 , 1:100.

Techniques: Expressing, Control

Journal: Brain Pathology

Article Title: Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

doi: 10.1111/bpa.13005

Figure Lengend Snippet: Quantification of GluN1 immunoreactivity within the CA1, CA2, CA3, dentate gyrus hippocampal subfields, subiculum, entorhinal cortex, and superior temporal gyrus in control and Alzheimer's disease groups. Data are expressed as mean with error bars representing SEM. The figure shows significant increases in GluN1 expression in specific layers of the hippocampal subfields (A–D) and the entorhinal cortex (F) in AD cases (white circles; n = 5–6) compared to controls (black circles; n = 6–7; unpaired Mann–Whitney test). AD, Alzheimer's disease; CA, cornu ammonis; DG, dentate gyrus; str. ori, straum oriens; str. pyr, stratum pyramidale; str. rad, stratum radiatum; str. mol, stratum moleculare; str. gran, stratum granulosum

Article Snippet: GluN1 , Recombinant protein corresponding to AA 660 to 811 from rat GluN1 , Synaptic Systems, Mouse, 114‐011, RRID:AB_887750 , 1:500 , 1:200 , 1:100.

Techniques: Control, Expressing, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N -Methyl- d -aspartate (NMDA) Receptors Adopt Different Subunit Arrangements *

doi: 10.1074/jbc.M113.469205

Figure Lengend Snippet: Arrangement of GluN1 subunits in GluN1/GluN2A NMDA receptors isolated from the total cellular pool. A , immunofluorescence analysis of protein expression. Cells expressing WT GluN1 plus FLAG/His 8 -GluN2A were fixed, permeabilized, and incubated with mouse monoclonal anti-GluN1 (ABD) and rabbit monoclonal anti-GluN2A primary antibodies followed by FITC-conjugated anti-mouse and Cy3-conjugated anti-rabbit secondary antibodies. Scale bar , 50 μm. B , schematic illustration of a GluN1/GluN2A NMDA receptor subunit heterodimer, with the location of the tag (FLAG/His 8 ) used to isolate the receptor indicated by the arrow , and the site of anti-GluN1 antibody decoration (NTD) indicated by the asterisk. Numbers refer to the subunits used (GluN1 and GluN2A). C , silver-stained gel of the isolated protein ( left ) showing the presence of bands at ∼120 and ∼180 kDa ( arrows ). Immunoblotting using anti-subunit antibodies indicated that these two bands were GluN1 and GluN2A, respectively ( center and right ). D , gallery of enlarged images showing receptors after incubation with anti-GluN1 (NTD) antibody. The gallery shows single- ( upper ) and double-decorated receptors ( lower ). Scale bar , 20 nm; height scale , 0–3 nm. E , frequency distribution of molecular volumes of the central particles decorated by anti-GluN1 (NTD) antibodies. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. F , frequency distribution of angles between pairs of bound antibodies. The subunit arrangement revealed by the data is shown in the inset. G , schematic illustration of a GluN1/GluN2A NMDA receptor subunit heterodimer, with the location of the tag (FLAG/His 8 ) used to isolate the receptor indicated by the arrow , and the site of anti-GluN1 antibody decoration (ABD) indicated by the asterisk. H , gallery of enlarged images showing receptors after incubation with anti-GluN1 (ABD) antibody. The gallery shows single- ( upper ) and double-decorated receptors ( lower ). Scale bar , 20 nm; height scale , 0–3 nm. I , frequency distribution of molecular volumes of the central particles decorated by anti-GluN1 (ABD) antibodies. J , frequency distribution of angles between pairs of bound antibodies. The subunit arrangement revealed by the data is shown in the inset. K , gallery of images of double-decorated receptors integrated into supported lipid bilayers and imaged under fluid. Bound antibodies are indicated by the arrowheads. Scale bar , 100 nm; height range , 20 nm.

Article Snippet: The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95, MAB2263, raised against an N-terminal peptide of rat GluA1), mouse monoclonal anti-GluN1 (Abcam; ab134308, S308-48, raised against amino acids 42–361 of GluN1), mouse monoclonal anti-GluN1 (Millipore; clone 54.1, MAB363, raised against amino acids 660–811 of GluN1), rabbit monoclonal anti-GluN2A (Millipore; clone A12W, 04-901, raised against residues 1265–1464 of mouse GluN2A), mouse monoclonal anti-Myc (Invitrogen; R950-25), mouse monoclonal anti-His (Fitzgerald; clone His-17, 10R-P134a), rabbit polyclonal anti-His (Fitzgerald; 70R-HR005), mouse monoclonal anti-V5 (Invitrogen; R960-25), mouse monoclonal anti-HA (Covance; HA.11 clone 16B12, MMS-101P), mouse monoclonal anti-FLAG (Sigma; clone M2, F3165), mouse monoclonal anti-β-actin (Sigma; clone AC-15, A5441), Cy3-conjugated goat anti-mouse (Sigma; C2181), Cy3-conjugated goat anti-rabbit (Sigma; C2306), and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771).

Techniques: Isolation, Immunofluorescence, Expressing, Incubation, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N -Methyl- d -aspartate (NMDA) Receptors Adopt Different Subunit Arrangements *

doi: 10.1074/jbc.M113.469205

Figure Lengend Snippet: Arrangement of GluN2A subunits in GluN1/GluN2A NMDA receptors isolated from the total cellular pool. A , schematic illustration of a GluN1/GluN2A NMDA receptor subunit heterodimer, with the location of the tag (FLAG/His 8 ) used to isolate the receptor indicated by the arrow , and the site of anti-His antibody decoration (post-TMD) indicated by the asterisk. Numbers refer to the subunits used (GluN1 and GluN2A). B , gallery of enlarged images showing receptors after incubation with anti-His antibody. The gallery shows single- ( upper ) and double-decorated receptors ( lower ). Scale bar , 20 nm; height scale , 0–3 nm. C , frequency distribution of molecular volumes of the central particles decorated by anti-His antibodies. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. D , frequency distribution of angles between pairs of bound antibodies. The subunit arrangement revealed by the data is shown in the inset .

Article Snippet: The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95, MAB2263, raised against an N-terminal peptide of rat GluA1), mouse monoclonal anti-GluN1 (Abcam; ab134308, S308-48, raised against amino acids 42–361 of GluN1), mouse monoclonal anti-GluN1 (Millipore; clone 54.1, MAB363, raised against amino acids 660–811 of GluN1), rabbit monoclonal anti-GluN2A (Millipore; clone A12W, 04-901, raised against residues 1265–1464 of mouse GluN2A), mouse monoclonal anti-Myc (Invitrogen; R950-25), mouse monoclonal anti-His (Fitzgerald; clone His-17, 10R-P134a), rabbit polyclonal anti-His (Fitzgerald; 70R-HR005), mouse monoclonal anti-V5 (Invitrogen; R960-25), mouse monoclonal anti-HA (Covance; HA.11 clone 16B12, MMS-101P), mouse monoclonal anti-FLAG (Sigma; clone M2, F3165), mouse monoclonal anti-β-actin (Sigma; clone AC-15, A5441), Cy3-conjugated goat anti-mouse (Sigma; C2181), Cy3-conjugated goat anti-rabbit (Sigma; C2306), and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771).

Techniques: Isolation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N -Methyl- d -aspartate (NMDA) Receptors Adopt Different Subunit Arrangements *

doi: 10.1074/jbc.M113.469205

Figure Lengend Snippet: Antibody decoration of additional epitope-tagged NMDA receptors isolated from the total cellular pool. A and B , schematic illustration of a GluN1/GluN2A NMDA receptor subunit heterodimer, with the location of the tag (HA/His 8 ) used to isolate the receptor indicated by the arrow , and the sites of antibody decoration ( A , anti-HA (ABD), and B , anti-GluN2A (CTD)) indicated by the asterisk. Numbers refer to the subunits used (GluN1 and GluN2A). C , frequency distribution of angles between pairs of bound anti-HA antibodies. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. D , frequency distribution of angles between pairs of bound anti-GluN2A antibodies. E and F , schematic illustration of a GluN1/GluN2A NMDA receptor subunit heterodimer, with the location of the tag (HA/His 8 ) used to isolate the receptor indicated by the arrow , and the sites of antibody decoration ( E , anti-Myc (ABD), and F , anti-HA (ABD)) indicated by the asterisk. G , frequency distribution of angles between pairs of bound anti-Myc antibodies. H , frequency distribution of angles between pairs of bound anti-HA antibodies. For each distribution, the subunit arrangement revealed by the data is shown in the inset .

Article Snippet: The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95, MAB2263, raised against an N-terminal peptide of rat GluA1), mouse monoclonal anti-GluN1 (Abcam; ab134308, S308-48, raised against amino acids 42–361 of GluN1), mouse monoclonal anti-GluN1 (Millipore; clone 54.1, MAB363, raised against amino acids 660–811 of GluN1), rabbit monoclonal anti-GluN2A (Millipore; clone A12W, 04-901, raised against residues 1265–1464 of mouse GluN2A), mouse monoclonal anti-Myc (Invitrogen; R950-25), mouse monoclonal anti-His (Fitzgerald; clone His-17, 10R-P134a), rabbit polyclonal anti-His (Fitzgerald; 70R-HR005), mouse monoclonal anti-V5 (Invitrogen; R960-25), mouse monoclonal anti-HA (Covance; HA.11 clone 16B12, MMS-101P), mouse monoclonal anti-FLAG (Sigma; clone M2, F3165), mouse monoclonal anti-β-actin (Sigma; clone AC-15, A5441), Cy3-conjugated goat anti-mouse (Sigma; C2181), Cy3-conjugated goat anti-rabbit (Sigma; C2306), and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771).

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N -Methyl- d -aspartate (NMDA) Receptors Adopt Different Subunit Arrangements *

doi: 10.1074/jbc.M113.469205

Figure Lengend Snippet: Subunit arrangement in cell surface GluN1/GluN2A NMDA receptors. A , protein eluted from monomeric avidin-agarose by biotin ( E1 ) and from anti-FLAG-agarose by triple-FLAG peptide ( E2 ) was analyzed by immunoblotting using anti-GluN1 ( left ), anti-GluN2A ( center ), or anti-β-actin antibodies ( right ). The right panel also shows an immunoblot of the total cell extract ( T ). B , gallery of enlarged images showing receptors double decorated by anti-GluN1 (ABD) antibodies. Scale bar , 20 nm; height scale , 0–3 nm. C , schematic illustration of a GluN1/GluN2A NMDA receptor subunit heterodimer, with the location of the tag (FLAG/His 8 ) used to isolate the receptor indicated by the arrow , and the site of anti-GluN1 antibody decoration (ABD) indicated by the asterisk. Numbers refer to the subunits used (GluN1 and GluN2A). D , frequency distribution of molecular volumes of the central particles decorated by anti-GluN1 antibodies. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. E , frequency distribution of angles between pairs of bound antibodies. The subunit arrangement revealed by the data is shown in the inset. F , schematic illustration of a GluN1/GluN2A NMDA receptor subunit heterodimer, with the location of the tag used to isolate the receptor indicated by the arrow , and the site of anti-His antibody decoration (post-TMD) indicated by the asterisk. G , frequency distribution of molecular volumes of the central particles decorated by anti-His antibodies. H , frequency distribution of angles between pairs of bound antibodies. The subunit arrangement revealed by the data is shown in the inset .

Article Snippet: The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95, MAB2263, raised against an N-terminal peptide of rat GluA1), mouse monoclonal anti-GluN1 (Abcam; ab134308, S308-48, raised against amino acids 42–361 of GluN1), mouse monoclonal anti-GluN1 (Millipore; clone 54.1, MAB363, raised against amino acids 660–811 of GluN1), rabbit monoclonal anti-GluN2A (Millipore; clone A12W, 04-901, raised against residues 1265–1464 of mouse GluN2A), mouse monoclonal anti-Myc (Invitrogen; R950-25), mouse monoclonal anti-His (Fitzgerald; clone His-17, 10R-P134a), rabbit polyclonal anti-His (Fitzgerald; 70R-HR005), mouse monoclonal anti-V5 (Invitrogen; R960-25), mouse monoclonal anti-HA (Covance; HA.11 clone 16B12, MMS-101P), mouse monoclonal anti-FLAG (Sigma; clone M2, F3165), mouse monoclonal anti-β-actin (Sigma; clone AC-15, A5441), Cy3-conjugated goat anti-mouse (Sigma; C2181), Cy3-conjugated goat anti-rabbit (Sigma; C2306), and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771).

Techniques: Avidin-Biotin Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N -Methyl- d -aspartate (NMDA) Receptors Adopt Different Subunit Arrangements *

doi: 10.1074/jbc.M113.469205

Figure Lengend Snippet: Demonstration of antibody specificity. tsA 201 cells expressing His /Myc-GluA2 ( A ), WT GluA1 ( B ), WT GluN1 ( C ), FLAG/His -GluN2A ( D ), HA/His -GluN1 ( E ), WT GluN2A ( F ), Myc-GluN1 ( G ), and HA/His -GluN2A ( H ) were fixed, permeabilized, and probed with monoclonal antibodies to the targets indicated above each panel , followed by appropriate Cy3-conjugated goat secondary antibody. Cells were imaged by confocal microscopy, and the fluorescence and brightfield channels were superimposed. Scale bar , 50 μm. The relevant figures in the paper are: A , ; B , and ; C , and ; D , and ; E–H , .

Article Snippet: The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95, MAB2263, raised against an N-terminal peptide of rat GluA1), mouse monoclonal anti-GluN1 (Abcam; ab134308, S308-48, raised against amino acids 42–361 of GluN1), mouse monoclonal anti-GluN1 (Millipore; clone 54.1, MAB363, raised against amino acids 660–811 of GluN1), rabbit monoclonal anti-GluN2A (Millipore; clone A12W, 04-901, raised against residues 1265–1464 of mouse GluN2A), mouse monoclonal anti-Myc (Invitrogen; R950-25), mouse monoclonal anti-His (Fitzgerald; clone His-17, 10R-P134a), rabbit polyclonal anti-His (Fitzgerald; 70R-HR005), mouse monoclonal anti-V5 (Invitrogen; R960-25), mouse monoclonal anti-HA (Covance; HA.11 clone 16B12, MMS-101P), mouse monoclonal anti-FLAG (Sigma; clone M2, F3165), mouse monoclonal anti-β-actin (Sigma; clone AC-15, A5441), Cy3-conjugated goat anti-mouse (Sigma; C2181), Cy3-conjugated goat anti-rabbit (Sigma; C2306), and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771).

Techniques: Expressing, Confocal Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N -Methyl- d -aspartate (NMDA) Receptors Adopt Different Subunit Arrangements *

doi: 10.1074/jbc.M113.469205

Figure Lengend Snippet: Quantitation of antibody decoration of AMPA and NMDA receptors

Article Snippet: The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95, MAB2263, raised against an N-terminal peptide of rat GluA1), mouse monoclonal anti-GluN1 (Abcam; ab134308, S308-48, raised against amino acids 42–361 of GluN1), mouse monoclonal anti-GluN1 (Millipore; clone 54.1, MAB363, raised against amino acids 660–811 of GluN1), rabbit monoclonal anti-GluN2A (Millipore; clone A12W, 04-901, raised against residues 1265–1464 of mouse GluN2A), mouse monoclonal anti-Myc (Invitrogen; R950-25), mouse monoclonal anti-His (Fitzgerald; clone His-17, 10R-P134a), rabbit polyclonal anti-His (Fitzgerald; 70R-HR005), mouse monoclonal anti-V5 (Invitrogen; R960-25), mouse monoclonal anti-HA (Covance; HA.11 clone 16B12, MMS-101P), mouse monoclonal anti-FLAG (Sigma; clone M2, F3165), mouse monoclonal anti-β-actin (Sigma; clone AC-15, A5441), Cy3-conjugated goat anti-mouse (Sigma; C2181), Cy3-conjugated goat anti-rabbit (Sigma; C2306), and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771).

Techniques: Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N -Methyl- d -aspartate (NMDA) Receptors Adopt Different Subunit Arrangements *

doi: 10.1074/jbc.M113.469205

Figure Lengend Snippet: Structure of partially dissociated GluN1/GluN2A NMDA receptors. A , examples of structures composed of four particles that are likely GluN1/GluN2A receptors that have attached to the mica intact and then partially dissociated. Adjacent small and large particles are indicated by arrowheads and arrows , respectively. Scale bar , 100 nm; height scale , 0–3 nm. B , frequency distribution of molecular volumes of individual small ( black bars ) and large particles ( gray bars ) within four-particle clusters. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated. C , three-dimensional representation of a partially dissociated NMDA receptor. The image is 80 nm square.

Article Snippet: The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95, MAB2263, raised against an N-terminal peptide of rat GluA1), mouse monoclonal anti-GluN1 (Abcam; ab134308, S308-48, raised against amino acids 42–361 of GluN1), mouse monoclonal anti-GluN1 (Millipore; clone 54.1, MAB363, raised against amino acids 660–811 of GluN1), rabbit monoclonal anti-GluN2A (Millipore; clone A12W, 04-901, raised against residues 1265–1464 of mouse GluN2A), mouse monoclonal anti-Myc (Invitrogen; R950-25), mouse monoclonal anti-His (Fitzgerald; clone His-17, 10R-P134a), rabbit polyclonal anti-His (Fitzgerald; 70R-HR005), mouse monoclonal anti-V5 (Invitrogen; R960-25), mouse monoclonal anti-HA (Covance; HA.11 clone 16B12, MMS-101P), mouse monoclonal anti-FLAG (Sigma; clone M2, F3165), mouse monoclonal anti-β-actin (Sigma; clone AC-15, A5441), Cy3-conjugated goat anti-mouse (Sigma; C2181), Cy3-conjugated goat anti-rabbit (Sigma; C2306), and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771).

Techniques:

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: TSG101 knockdown selectively alters glutamate receptor surface expression. (A) Representative immunoblots showing total and surface-biotinylated NMDA receptor subunits GluN1 and GluN2A in HEK293 cells transfected with scramble shRNA (control) or shTSG101. Surface proteins were isolated by sulfo-NHS-SS-biotin-based biotinylation and analysed by immunoblotting. GAPDH served as a loading control for total lysate fractions and was absent from biotinylated fractions. (B–C) Quantification of surface GluN1 (B) and GluN2A (C) normalised to total receptor levels and expressed as fold change relative to scramble shRNA. TSG101 knockdown significantly increased the surface abundance of both NMDA receptor subunits. (D) Representative immunoblots showing total and surface-biotinylated AMPA receptor subunits GluA1 and GluA2. (E–F) Quantification of surface GluA1 (E) and GluA2 (F) normalised to total receptor levels. TSG101 knockdown increased GluA1 surface expression; GluA2 surface levels were not significantly altered. (G) Representative immunoblots showing total and surface-biotinylated GluK2. (H) Quantification of GluK2 surface expression normalised to total protein, showing increased surface abundance following TSG101 knockdown. Data are presented as mean ± SEM (n = 3 independent experiments). Unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Knockdown, Expressing, Western Blot, Transfection, shRNA, Control, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: ESCRT modulation alters surface localisation of glutamate receptor subunits in HEK293 cells. (A) Representative immunocytochemical images showing surface staining of GluN1 (red) and GluN2A (green) in HEK293 cells under control conditions, following shTSG101, or expression of dominant-negative VPS4a (DN-VPS4a). Merged images include nuclear staining (blue). Scale bar: 20 µm. (B) Quantification of surface GluN1 and GluN2A fluorescence intensity normalised to control. Both shTSG101 and DN-VPS4a significantly altered surface expression of GluN1 and GluN2A. (C–E) Representative images of surface GluA1 (C), GluA2 (D), and GluK2 (E) staining under control, shTSG101, and DN-VPS4a conditions. Scale bars: 20 µm. (F) Quantification of surface GluA1 fluorescence intensity. Both shTSG101 and DN-VPS4a significantly increased GluA1 surface expression. (G) Quantification of surface GluA2 fluorescence intensity. Surface GluA2 showed modest or non-significant changes depending on condition. (H) Quantification of surface GluK2 fluorescence intensity. Both shTSG101 and DN-VPS4a significantly altered GluK2 surface expression. Data are presented as mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Staining, Control, Expressing, Dominant Negative Mutation, Fluorescence

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: NMDA receptor surface expression increases upon ESCRT pathway disruption in primary cortical neurons. (A–C) Representative immunoblots and quantification showing increased surface/total ratios of GluN1 and GluN2A in primary cortical neurons (DIV14) following shTSG101 transduction. β-actin served as a loading control. (D–F) Representative immunoblots and quantification showing increased GluN1 and GluN2A surface/total ratios following DN-VPS4a expression. (G–H) Representative immunocytochemical images and quantification of surface GluN1 and GluN2A under non-permeabilised conditions in neurons expressing scramble shRNA (control) or shTSG101. EGFP indicates transduction (green). Scale bars: 20 µm. (I–L) Representative immunocytochemical images and quantification of surface GluN1 and GluN2A in neurons expressing control vector or DN-VPS4a. Scale bars: 20 µm. Data are presented as mean ± SEM. For biochemical assays, n = 3 independent experiments; for immunocytochemistry, n = 6–8 neurons per condition from 3 independent cultures. One-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Expressing, Disruption, Western Blot, Transduction, Control, shRNA, Plasmid Preparation, Immunocytochemistry

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: GluN1 redistributes across early, recycling, and late endosomes following ESCRT disruption in primary hippocampal neurons. (A–C) Representative dendritic images showing GluN1 (green) and the early endosome marker EEA1 (red) in control neurons (A), shTSG101-expressing neurons (B), and DN-VPS4a-expressing neurons (C). Merged images with single-channel views and line-scan intensity profiles of GluN1 and EEA1 along dendrites are shown below each condition. Scale bars: 20 µm (overview), 5 µm (insets). (D–F) Representative dendritic images showing GluN1 (green) and the recycling endosome marker Rab11 (red) in control neurons (D), shTSG101-expressing neurons (E), and DN-VPS4a-expressing neurons (F). Line-scan intensity profiles are shown below each condition. Scale bars: 20 µm (overview), 5 µm (insets). (G–I) Representative dendritic images showing GluN1 (green) and the late endosome marker Rab7 (red) in control neurons (G), shTSG101-expressing neurons (H), and DN-VPS4a-expressing neurons (I). Line-scan intensity profiles are shown below each condition. Scale bars: 20 µm (overview), 5 µm (insets). (J–L) Quantification of Pearson’s correlation coefficient (R) for GluN1 colocalisation with EEA1 (J), Rab11 (K), and Rab7 (L) under control, shTSG101, and DN-VPS4a conditions. (M–O) Quantification of Manders’ tM2 values representing the fraction of GluN1 signal within EEA1-positive (M), Rab11-positive (N), and Rab7-positive (O) endosomes. (P) Quantitative summary table of GluN1 colocalisation changes with early (EEA1), recycling (Rab11), and late (Rab7) endosomal markers following shTSG101 or DN-VPS4a. Data represent 3 biological replicates, n = 6 dendrites per condition. All data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ****p < 0.0001; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Disruption, Marker, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Implication of N -Methyl- d -Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration

doi: 10.3390/ijms22179356

Figure Lengend Snippet: Evaluation of retinal epithelial cells (RPE) NMDAR expression in both in vivo and in vitro models of HHcy. ( a ) RT-qPCR analysis showing the expression of the NMDAR subunit NR1 in the human RPE (ARPE-19) cell line as compared to human neuroblastoma cells (ATCC CRL-2266) used as a positive control. ( b ) RT-qPCR analysis confirming NMDA receptor subunits NR1 (120 kD) in ARPE-19 cells and its activation by Hcy treatment (20 and 50 µM) as compared control untreated cells. ( c ) Western blot analysis showing the expression of the NMDAR subunit NR1 in human RPE (ARPE-19) treated with different concentrations of Hcy (20, 50, and 100 µM Hcy). GADPH was used as a loading control. ( d ) IF analysis showing the increased expression of NMDAR1 (green) in ARPE-19 treated with different concentrations of Hcy (20, 50, and 100 µM Hcy). ( e ) Western blot analysis showing the expression of NMDAR in the outer retina (containing mainly RPE cells) of the WT mice and cbs +/− mice. GADPH was used as a loading control. ( f ) IF analysis showing the increased expression of NMDAR1 (green) in primary RPE cells isolated from cbs +/− mice ( n = 6 mice per group) for cells ( n = 4). Calibration bar: 50 μm; * p < 0.05 and ** p < 0.01.

Article Snippet: Samples were subsequently subjected to gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and the protein was blotted onto nitrocellulose membranes, which were further blocked using a 5% milk solution and then incubated with the following antibodies: NMDAR1 (Cell signaling, Danvers, MA, USA, Ca # 5704S), NMDAR2A (Cell signaling, Danvers, MA, USA, Ca # 4205s), NMDAR2B (Cell signaling, Danvers, MA.

Techniques: Expressing, In Vivo, In Vitro, Quantitative RT-PCR, Positive Control, Activation Assay, Control, Western Blot, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Implication of N -Methyl- d -Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration

doi: 10.3390/ijms22179356

Figure Lengend Snippet: Deletion of NMDAR in RPE cells decreased Hcy-induced CNV in retinal flat mounts. ( a ) Immunofluorescence staining for RPE flat-mounts isolated from mouse retinas after one week of intravitreal injection of Hcy in WT and NMDAR R −/− as compared to WT control non-injected mice and mice with HHcy ( cbs +/− ) stained with a vascular marker using Isolectin-B4 (red) and NMDAR1 (green). Hcy injection induced choroidal neovascularization and activation of NMDAR, which was more evident in both mice models of HHcy ( cbs +/− mice and Hcy-injected WT mice), while knocking down NMDAR in ( NMDAR R −/− ) was able to reduce CNV induction and NMDAR activation by Hcy injection. ( b ) OCT images and Insight ® software were used for assessment of the thickness of different retinal layers in wild-type and NMDAR R −/− mice 72 h after intravitreal injection of Hcy. ( c ) Analysis of retinal thickness of WT mice and NMDAR R −/− mice injected with Hcy showed an improved RPE layer and decreased CNV size in NMDAR R −/− mice. ( d ) Outer retina flat-mounts stained with an antibody for NMDAR (green), confirming that both the pharmacological (MK801) and the genetic inhibition of NMDAR were able to block the Hcy activation of NMDAR. ( n = 6 mice per group). Calibration bar: 20 µm; * p < 0.05, ** p < 0.01.

Article Snippet: Samples were subsequently subjected to gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and the protein was blotted onto nitrocellulose membranes, which were further blocked using a 5% milk solution and then incubated with the following antibodies: NMDAR1 (Cell signaling, Danvers, MA, USA, Ca # 5704S), NMDAR2A (Cell signaling, Danvers, MA, USA, Ca # 4205s), NMDAR2B (Cell signaling, Danvers, MA.

Techniques: Immunofluorescence, Staining, Isolation, Injection, Control, Marker, Activation Assay, Software, Inhibition, Blocking Assay

Journal: JCI Insight

Article Title: Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons

doi: 10.1172/jci.insight.127736

Figure Lengend Snippet: (A) Immunofluorescence images showing NR1+ punctae on neurites of iMNs treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. Scale bar: 2 μm. This experiment was repeated 3 times with similar results. (B and C) NR1+ punctae per unit area in control, C9-ALS (B), or sporadic ALS (C) iMNs. Each gray circle represents the number of NR1+ punctae per area unit on a single neurite (1 neurite quantified per iMN). n = 33 (controls and C9-ALS) or 13 (sporadic) iMNs quantified per line per condition from 2 biologically independent iMN conversions of 2 CTRL, 2 C9-ALS, or 6 sporadic ALS lines. Median ± interquartile range. Kruskal-Wallis testing. (D) Number of calcium transients per 30 seconds in control or C9-ALS iMNs treated with 10 nM inactive 3K3A-APC or 3K3A-APC. n = 21 iMNs per line per condition from 3 biologically independent iMN conversions of 3 CTRL and 3 C9-ALS lines. For the C9-ALS plus 3K3A-APC condition, n = 19 iMNs per line. Median ± interquartile range. Kruskal-Wallis testing. (E) Number of calcium transients per 30 seconds in control or sporadic ALS iMNs treated with inactive 3K3A-APC or 3K3A-APC. n = 20 iMNs per line per condition from 3 biologically independent iMN conversions of 3 CTRL and 1 sporadic line. Median ± interquartile range. Kruskal-Wallis testing. (F) Immunoblotting of surface NR1 after surface protein biotinylation in C9-ALS iMNs generated with NGN2, ISL1, and LHX3 and treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. (G) Quantification of NR1 immunoblotting from F. n = 4 biologically independent iMN conversions. Each gray circle represents an individual sample. The ratio of surface to total transferrin receptor was used to normalize for the membrane protein extraction efficiency and TUJ1 was used to normalize for neuron number. (H) Immunoblotting of surface NR1 after surface protein biotinylation in sporadic ALS iMNs (1 patient) generated with NGN2, ISL1, and LHX3 and treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. The full blot for total TUJ1 is shown. (I) Quantification of NR1 immunoblotting from H. n = 4 biologically independent iMN conversions. Each gray circle represents an individual sample. The ratio of surface to total transferrin receptor was used to normalize for the membrane protein extraction efficiency and TUJ1 was used to normalize for neuron number. The day of differentiation stated on each panel indicates the day of differentiation on which the experimental treatment or time course was initiated. TF, transferrin.

Article Snippet: After snap freezing, tissue was sectioned by cryostat at 20-μm thickness and stained with the following primary antibodies: anti-poly(GP) (catalog 24494-1-AP, ProteinTech), anti-poly(GR) (catalog MABN778, Millipore), anti-poly(PR) (catalog 23979-1-AP, ProteinTech), anti-NR1 (catalog NB300118, Novus), and anti-MAP2 (catalog ab5392, Abcam).

Techniques: Immunofluorescence, Western Blot, Generated, Protein Extraction

Journal: JCI Insight

Article Title: Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons

doi: 10.1172/jci.insight.127736

Figure Lengend Snippet: (A) Overview of the experimental procedure for testing the ability of 3K3A-APC to reduce DPR levels in the hippocampus of C9-BAC mice. (B–D) The effect of 10 nM inactive 3K3A-APC or 3K3A-APC on the level of poly(GR)+ punctae in the dentate gyrus of C9-BAC mice. Mean ± SD of the number of poly(GR)+ (B), poly(GP)+ (C), and poly(PR)+ (D) punctae per cell; each data point represents a single cell. Cells quantified from 3 mice per condition, 1-way ANOVA with Tukey’s correction for all comparisons. Scale bars: 10 μm. Dotted lines outline cell bodies. Neuronal area was determined by manual outlining in ImageJ on the basis of the staining pattern provided by TUJ1 or MAP2. (E) Overview of the experimental procedure for inducing NMDA injury in the hippocampus and testing the ability of 3K3A-APC to mitigate this injury. (F and G) The effect of 0.2 μg of 3K3A-APC delivered in a volume of 0.3 μL on NMDA-induced hippocampal injury in C9orf72+/+ and C9orf72+/– mice. Mean ± SEM of n = 3 mice per condition, 1-way ANOVA with Tukey’s correction across all comparisons. Red dashed lines outline the injury sites (F). Vehicle control conditions were published in a previous study (4). (H and I) Immunostaining (H) and quantification (I) of NR1 levels in C9orf72+/– mice treated with vehicle or 0.2 μg of 3K3A-APC delivered in a volume of 0.3 μL (n = 3 mice per condition, 72 cells quantified per condition). Each gray data point represents a single cell. Mean ± interquartile range. Mann-Whitney test.

Article Snippet: After snap freezing, tissue was sectioned by cryostat at 20-μm thickness and stained with the following primary antibodies: anti-poly(GP) (catalog 24494-1-AP, ProteinTech), anti-poly(GR) (catalog MABN778, Millipore), anti-poly(PR) (catalog 23979-1-AP, ProteinTech), anti-NR1 (catalog NB300118, Novus), and anti-MAP2 (catalog ab5392, Abcam).

Techniques: Staining, Immunostaining, MANN-WHITNEY

Journal: Bioengineering

Article Title: A Bivalent Protease-Activated Receptor-Derived Peptide Mimics Neuronal Anti-Apoptotic Activity of Activated Protein C

doi: 10.3390/bioengineering12090899

Figure Lengend Snippet: Analysis of primary cultured mouse cortical neurons after seven days in culture. ( A ) Neurons stained with MAP2 (green), nuclear stain DAPI (blue), and neuronal nuclear antigen (NeuN, red). DAPI-positive nuclei were on average 94% positive for NeuN neuronal marker in cultures (graph, mean ± SD, from three independent cultures). ( B ) Western blot analyses of NMDA receptor subunits NR1, NR2A, NR2B, NR2C, NR2D, and NR3A from three independent cultures with each lane representing an independent culture.

Article Snippet: A rabbit monoclonal anti-NR1 antibody (catalog number A11699) was purchased from Abclonal (Woburn, MA, USA).

Techniques: Cell Culture, Staining, Marker, Western Blot

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 1. CADD scores and protein modelling predict stronger functional effects for EAS-associated GRIN2A mutations than in controls. (a) Protein structure model of NMDAR (PDB ID 4TLL): GluN1 (grey and green), GluN2 (B in this structure) (blue and red). Membrane would be horizontal in this image with the NTD and ABD in the extracellular space. Intracellular C-Terminal domain would be below the transmembrane domain (not present in this structure). (b) Schematic linear representation of GluN2A with the domains annotated. Black rectangles indicate transmembrane domains. Plot of scaled CADD scores against GluN2A amino acid position for missense variants. Black dots represent scores for 65/6474 individuals from the Exome Variant Server (EVS) that had missense variants in GRIN2A and coloured symbols are scores for variants found in individuals with EAS disorders. The horizontal dotted line indicates the scaled CADD score cut off of 20 for a highly likely deleterious variant. (c) Protein structure model of the NTD of NMDAR (PDB ID 3QEL): GluN1 (grey), GluN2 (B in this structure) (red). Mutations considered in this domain highlighted (conserved between GluN2A and B). (d) Protein structure model of the LBD of NMDAR (PDB ID 2A5T): GluN1 (grey), GluN2A (blue). Mutations considered in this paper highlighted, as well as agonists.

Article Snippet: Human GRIN1 (GenBank accession NM_000832.5) cDNA clone was also purchased from Origene Technologies (Cat#: SC308819) and inserted into a second pcDNA3.1 (+) vector.

Techniques: Functional Assay, Membrane, Variant Assay

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 5. GRIN2A mutations reduce total protein levels and membrane trafficking of GluN2A. (a) Representative Western blot of HEK lysates probed with anti-GluN2A antibody (top), and anti-GAPDH (bottom) as a loading control. Bands around 180 kDa indicate GluN2A. WT, wild type, UT – untransfected. Right is Amersham Full-Range rainbow molecular weight marker with the blot imaged in visible light. (b) Plot of amount of GluN2A protein, normalised to WT, from Western blotting of total cell lysates of transiently co- transfected HEK cells 48-hours post transfection. Average of 3 blots from 3 independent transfections. Error bars indicated SEM. (c) Fixed and immunolabelled co-transfected HEK cells with anti-HA antibody (red) to detect surface GluN2A expression, and fixed, permeabilised and immunolabelled with the same antibody to detect total GluN2A protein levels. Scale bar 25 µm. Nuclei stained with Hoechst (blue). (d) Quantitation of surface and total GluN2A protein levels, averaged over the total number of cells analyzed for each condition (n is between 903 to 2255 cells), reveals greatly reduced surface expression of GluN2A as measured by fluorescence intensity. Dunnett’s corrected one-way ANOVA for membrane or total intensity as compared to WT *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant. Average ± SEM. (e) Relative surface levels of NMDARs correlated with the log of glutamate EC50 (Pearson’s coefficient of determination r2 = 0.77, two-tailed p = 0.002). (f) Normalised CRC from single-cell calcium-flux imaging. Response to increasing concentrations of glutamate from HEK cells co-transfected with decreasing quantities of WT GRIN2A per well (100% = 320 ng) with standard 320 ng GRIN1 per well. Response is compared to mutant P79R. Error bars ± SEM.

Article Snippet: Human GRIN1 (GenBank accession NM_000832.5) cDNA clone was also purchased from Origene Technologies (Cat#: SC308819) and inserted into a second pcDNA3.1 (+) vector.

Techniques: Membrane, Western Blot, Control, Molecular Weight, Marker, Transfection, Expressing, Staining, Quantitation Assay, Fluorescence, Two Tailed Test, Imaging, Mutagenesis

Journal:

Article Title: N-Methyl-D-Aspartate receptor subunit phenotypes of vagal afferent neurons in nodose ganglia of the rat

doi: 10.1002/cne.20955

Figure Lengend Snippet: Primary antibodies

Article Snippet: NR1 , Santa Cruz, CA , sc-1467 , Goat polyclonal , 1:100 , NR1 subunit carboxyl terminus, amino acids 918–938 , Single 115kDa band for pan-NR1 ( Marvizon et al., 2002 ; SC).

Techniques: Sequencing, Western Blot

Journal:

Article Title: N-Methyl-D-Aspartate receptor subunit phenotypes of vagal afferent neurons in nodose ganglia of the rat

doi: 10.1002/cne.20955

Figure Lengend Snippet: Photomicrographs of nodose ganglion neurons labeled with affinity-purified polyclonal primary antisera raised in goat against the NR1 (A), NR2B (B), NR2C (C), and NR2D (D) NMDA receptor subunits. Panels on the left (A, C) show stained neurons in longitudinal sections (20 μm) from the cranial (A,C) and caudal (B,D) portions of nodose ganglia. Scale bar = 50 μm.

Article Snippet: NR1 , Santa Cruz, CA , sc-1467 , Goat polyclonal , 1:100 , NR1 subunit carboxyl terminus, amino acids 918–938 , Single 115kDa band for pan-NR1 ( Marvizon et al., 2002 ; SC).

Techniques: Labeling, Affinity Purification, Staining

Journal:

Article Title: N-Methyl-D-Aspartate receptor subunit phenotypes of vagal afferent neurons in nodose ganglia of the rat

doi: 10.1002/cne.20955

Figure Lengend Snippet: Longitudinal serial sections from the same nodose ganglion, separated by 40 μm, stained with affinity-purified polyclonal primary antisera raised in goat against the NR1 (A), and NR2D (B) NMDA receptor subunits. Note that NR1 and NR 2D-labeled neurons are fairly evenly distributed throughout the ganglia, whereas in some instances NR2C-IR perikarya were gathered into clusters of cells (C). Scale bar = 200 μm.

Article Snippet: NR1 , Santa Cruz, CA , sc-1467 , Goat polyclonal , 1:100 , NR1 subunit carboxyl terminus, amino acids 918–938 , Single 115kDa band for pan-NR1 ( Marvizon et al., 2002 ; SC).

Techniques: Staining, Affinity Purification, Labeling

Journal: EMBO Reports

Article Title: The DNA repair protein DNA-PKcs modulates synaptic plasticity via PSD-95 phosphorylation and stability

doi: 10.1038/s44319-024-00198-3

Figure Lengend Snippet: ( A ) Representative Western Blot analysis showing the expression of DNA-PKcs protein in different mouse brain regions at similar levels and in mouse primary cortical neurons. HEK293 cells were used as positive control. α-Tubulin was used as a loading control. ( B ) Western Blots representing the biochemical fractionation of mouse cortical neurons (DIV 21) showing the synaptic localization (LP1) of DNA-PKcs. Equal amounts of protein (70 μg) were loaded for each fraction ( n = 3 independent experiments). Fractions were loaded on the gel and the specificity of the fractionation procedure was confirmed by using specific markers for subcellular compartments: Lamin A/C for the nuclear fraction (P1), PSD-95 for the synaptosomal membrane fraction (LP1) and Synapsin I for the synaptic vesicle fraction (LP2). Histogram data represent the percentage with respect to a total protein extract of DNA-PKcs in the different cellular fractions. Results are expressed as mean ± SEM. ( C ) Representative confocal fluorescence images of mouse primary cortical neurons (DIV 21) labeled with the anti-DNA-PKcs antibody (green channel), the neuronal marker MAP2 (red channel), and DNA dye (blue, DAPI). DNA-PKcs is strongly expressed in neurons and is distributed both in the cell soma and dendrites. DNA-PKcs antibody specificity is confirmed by the absence of immunofluorescence when the antibody is preincubated with an excess of recombinant DNA-PKcs protein (lower panel). Scale Bar 5 μm. ( D ) Representative triple immunofluorescence THUNDER images of mouse primary cortical neurons (DIV 21) showing the distribution of DNA-PKcs (green channel) similar to synaptic proteins such as Synapsin I, Syntaxin I, PSD-95, GluN1, and GluA1 (red channel). Scale Bar 5 μm. Images of neurites from a single neuron show the punctate co-localization of DNA-PKcs with pre- and postsynaptic proteins. Single-channel images are provided to better evaluate the localization of each protein. Insets represent enlargements of a dendritic tract showing DNA-PKcs co-localization with pre- and postsynaptic proteins. White dotted edges in the red channels highlight the position of DNA-PKcs protein with respect to synaptic proteins. Scale Bar 2 μm. ( E ) Histogram data represent the percentage of DNA-PKcs puncta co-localization with the different synaptic proteins analyzed. Results are expressed as mean ± SEM; n = 3 independent experiments. Data distribution is shown in the enlargement; n = 21 neurons/each synaptic marker. ( F ) DNA-PKcs kinase activity assay performed using human and mouse cortical membrane fractions. Protein extracts from HEK293 cells (200 μg), LP1 human cortex fraction (200 μg), and mouse LP1 fraction (1 mg) were subject to DNA-PKcs immunoprecipitation and the phosphorylation assay was performed. The assay performed without protein extract was used as a negative control. Data were expressed as ratio of values in presence/absence of the DNA-PKcs substrate p53 (presented as mean values ± SEM of kinase activity; n = 3 independent experiments). P1, nuclei and large debris; S3, cytosolic fraction; P3, light membrane fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction; LS2, supernatant from LP2; IP, immunoprecipitated. .

Article Snippet: The phosphorylation of seven different proteins by DNA-PKcs was evaluated: DNA-PKcs LOT002, (Invitrogen product PR9107A, Lot#1484325 A), ERK2, active (ProQinase product #0634-0000-7, Lot#008), ERK2, non-activated (ProQinase product #0634-0000-1, Lot#005), RPS6KA1 LOT002, (Invitrogen product PV4049, Lot#386267Z1A), DLG4 (Origene product TP315178, Lot#060811), GRIN1 (Origene product TP319368, Lot#100814) TP53 (Origene product TP300003, Lot#17J10L19), and GRIA1 (Origene product TP326253, Lot#BJ0A64E).

Techniques: Western Blot, Expressing, Positive Control, Control, Fractionation, Membrane, Fluorescence, Labeling, Marker, Immunofluorescence, Recombinant, Kinase Assay, Immunoprecipitation, Phospho-proteomics, Negative Control, Activity Assay

Journal: EMBO Reports

Article Title: The DNA repair protein DNA-PKcs modulates synaptic plasticity via PSD-95 phosphorylation and stability

doi: 10.1038/s44319-024-00198-3

Figure Lengend Snippet: Representative triple immunofluorescence THUNDER images of mouse primary cortical neurons (DIV 21) labeled with the anti-DNA-PKcs antibody (green channel), the synaptic markers: Synapsin I, Syntaxin I, PSD-95, GluN1, and GluA1 (red channel), one at a time, and the neuronal marker MAP2 (blue). Single-channel images are provided to better show the localization of each synaptic marker and the distribution of DNA-PKcs similar to the synaptic proteins. Scale Bar 5 μm.

Article Snippet: The phosphorylation of seven different proteins by DNA-PKcs was evaluated: DNA-PKcs LOT002, (Invitrogen product PR9107A, Lot#1484325 A), ERK2, active (ProQinase product #0634-0000-7, Lot#008), ERK2, non-activated (ProQinase product #0634-0000-1, Lot#005), RPS6KA1 LOT002, (Invitrogen product PV4049, Lot#386267Z1A), DLG4 (Origene product TP315178, Lot#060811), GRIN1 (Origene product TP319368, Lot#100814) TP53 (Origene product TP300003, Lot#17J10L19), and GRIA1 (Origene product TP326253, Lot#BJ0A64E).

Techniques: Immunofluorescence, Labeling, Marker

Journal: EMBO Reports

Article Title: The DNA repair protein DNA-PKcs modulates synaptic plasticity via PSD-95 phosphorylation and stability

doi: 10.1038/s44319-024-00198-3

Figure Lengend Snippet: ( A ) Immunofluorescence images of mouse primary cortical neurons (DIV 9), labeled with an anti-DNA-PKcs antibody, unstimulated (DMSO), or after chemical LTP induced by forskolin/rolipram stimulation (F/R). F/R treatment induces an increase of DNA-PKcs protein levels in neurites proximal to the cell body (red arrows). Histogram data represent the mean intensity of DNA-PKcs immunolabelling in DMSO and F/R treated cells. Bars in the plots represent means ± SEM ( n = 3 independent experiments, * p < 0.05 Statistics by Student’s t -test). Data distribution is shown in the enlargement; n = 15 neurons/each treatment. Scale Bar 5 μm. ( B ) Western Blot showing no increase of DNA-PKcs in total protein extract from primary cortical neurons after F/R stimulation. Vinculin was used as a loading control. ( C ) Representative Western Blot showing the biochemical fractionation of mouse primary cortical neurons (DIV 9) unstimulated (DMSO) or after F/R treatment. Following F/R stimulation, DNA-PKcs is enriched in the synaptosomal membrane fraction (LP1). Vinculin was used as a loading control. Histogram data represent the percent change in DNA-PKcs band intensity in P3 and LP1 (gray background) fractions in F/R stimulated cells with respect to DMSO-treated cultures. Statistics by Student’s t -test (unpaired, two-tailed) for F/R vs DMSO in P3 fraction ( p = 0.241; n = 3 independent experiments) and for F/R vs DMSO in LP1 fraction (* p < 0.05; n = 3 independent experiments). Bars in the plots represent means ± SEM. ( D ) Immunoprecipitation of DNA-PKcs from LP1 (500 μg) mouse cortex. Western Blot analysis using the anti-DNA-PKcs antibody shows the full-length protein in the Input and IP lanes. IgG control antibody isotype is also shown ( n = 3 independent experiments). ( E ) Immunoprecipitation of DNA-PKcs from LP1 mouse cortex. Western Blot analysis using anti-PSD-95, anti-GluN1, and anti-GluN2A/B antibodies indicates that DNA-PKcs pulls down the three postsynaptic proteins (IP lanes). IgG were loaded as non-specific antibody IP control ( n = 3 independent experiments). .

Article Snippet: The phosphorylation of seven different proteins by DNA-PKcs was evaluated: DNA-PKcs LOT002, (Invitrogen product PR9107A, Lot#1484325 A), ERK2, active (ProQinase product #0634-0000-7, Lot#008), ERK2, non-activated (ProQinase product #0634-0000-1, Lot#005), RPS6KA1 LOT002, (Invitrogen product PV4049, Lot#386267Z1A), DLG4 (Origene product TP315178, Lot#060811), GRIN1 (Origene product TP319368, Lot#100814) TP53 (Origene product TP300003, Lot#17J10L19), and GRIA1 (Origene product TP326253, Lot#BJ0A64E).

Techniques: Immunofluorescence, Labeling, Western Blot, Control, Fractionation, Membrane, Two Tailed Test, Immunoprecipitation

Journal: EMBO Reports

Article Title: The DNA repair protein DNA-PKcs modulates synaptic plasticity via PSD-95 phosphorylation and stability

doi: 10.1038/s44319-024-00198-3

Figure Lengend Snippet: ( A ) Western blots of total (Tot) and synaptosomal membrane extracts (LP1) from WT and DNA-PKcs −/− cortex and hippocampus demonstrating reduction of postsynaptic proteins specifically in LP1 fractions of mutant mice. Histogram data for the cortex and hippocampus represent the normalized percent change in protein band intensity in DNA-PKcs −/− mice with respect to WT (100%). (PSD-95 in LP1 from cortex: WT: 100 ± 5.2%, DNA-PKcs −/−: 42.5 ± 3.5%; * p < 0.05; GluN1 in LP1 from cortex: WT: 100 ± 3.9%, DNA-PKcs −/−: 59.5 ± 3.2%; * p < 0.05; GluA1 in LP1 from cortex: WT: 100 ± 8.2%, DNA-PKcs −/−: 60 ± 4.2%; * p < 0.05; GluN2A in LP1 from cortex: WT: 100 ± 5.1%, DNA-PKcs −/−: 66.7 ± 5.7%; * p < 0.05); (PSD-95 in LP1 from hippocampus: WT: 100 ± 5.1%, DNA-PKcs −/−: 41.8 ± 3.4%; * p < 0.05; GluN1 in LP1 from hippocampus: WT: 100 ± 5.8%, DNA-PKcs −/−: 56.4 ± 4.5%; * p < 0.05; GluA1 in LP1 from hippocampus: WT: 100 ± 5%, DNA-PKcs −/−: 63 ± 3.9%; * p < 0.05; GluN2A in LP1 from hippocampus: WT: 100 ± 5.8%, DNA-PKcs −/−: 70 ± 2.9%; * p < 0.05). α-Tubulin was used as a loading control. Results are expressed as mean ± SEM. Statistics by Student’s t -test (unpaired, two-tailed) ( n = 3 mice). ( B ) Upper panel, fluorescence THUNDER images of cortical neurons (DIV 21) from WT and DNA-PKcs −/− mice, triple labeled with anti-PSD-95 antibody (green channel), Synaptophysin I (red) and DNA dye (blue, DAPI). Lower panel, triple fluorescence images of primary cortical neurons (DIV 21) from WT and DNA-PKcs −/− mice, labeled with anti-PSD-95 and anti-MAP2 (red channel) antibodies. Scale Bar 5 μm. High-magnification images of neurites confirm the reduction of PSD-95 staining in mutant neurons. Scale Bar 2 μm. Histogram data represent mean intensity ± SEM of fluorescence of PSD-95 labeling, plotted as a percentage of control and show a significant reduction of PSD-95 protein in mutant neurons. Statistics by Student’s t -test (unpaired, two-tailed) ( n = 3 independent cultures, * p < 0.05). Data distribution is shown in the enlargement; WT n = 33 neurons, DNA-PKcs −/− n = 36 neurons. ( C ) Immunofluorescence THUNDER images of primary cortical neurons (DIV 21) from WT and DNA-PKcs −/− mice, labeled with anti-GluA1 (green channel), anti-MAP2 (red channel) antibodies and DNA dye (blue, DAPI). High-magnification images of neurites confirm the reduction of GluA1 staining in mutant neurons. Scale Bar 2 μm. Histogram data represent mean intensity ± SEM of fluorescence of GluA1 labeling, plotted as percentage of control, and show a significant reduction of GluA1 expression in DNA-PKcs −/− neurons. Statistics by Student’s t -test (unpaired, two-tailed) ( n = 3 independent cultures, * p < 0.05). Scale Bar 5 μm. Data distribution is shown in the enlargement; WT n = 39 neurons, DNA-PKcs −/− n = 36 neurons. ( D ) Western blot analysis of synaptosomal membranes purified from WT and DNA-PKcs −/− neurons unstimulated (DMSO) or after F/R stimulation. Although synaptosomal membranes from DNA-PKcs −/− neurons have a lower expression of the GluA1 receptor subunit, after chemical stimulation, GluA1 incorporation in DNA-PKcs −/− synapses is as efficient as in WT. Band intensities, quantified and normalized by PSD-95, are expressed as fold change. α-Tubulin was used as a loading control. Error bars in histograms indicate SEM. Statistics by Student’s t -test (unpaired, two-tailed) ( n = 4 independent experiments, p = 0.1579). ( E ) Representative Western blot analysis of surface and total levels of GluA1 (assessed using cell-surface biotinylation) on hippocampal slices at T0 or 40 min after HFS delivery in WT and DNA-PKcs −/− mice. The levels of surface GluA1 remain constant up to 40 min following HFS in DNA-PKcs −/− slices (1.07 ± 0.055 folds change; p = 0.3791) as compared with WT slices that show an increased level of surface GluA1 after HFS (2.51 ± 0.2 folds change; p < 0.01). The surface-to-total ratio was calculated and expressed as mean ± SEM. Statistics by Student’s t -test (unpaired, two-tailed) ( n = 3 independent slices per each experimental group, * p < 0.05). .

Article Snippet: The phosphorylation of seven different proteins by DNA-PKcs was evaluated: DNA-PKcs LOT002, (Invitrogen product PR9107A, Lot#1484325 A), ERK2, active (ProQinase product #0634-0000-7, Lot#008), ERK2, non-activated (ProQinase product #0634-0000-1, Lot#005), RPS6KA1 LOT002, (Invitrogen product PV4049, Lot#386267Z1A), DLG4 (Origene product TP315178, Lot#060811), GRIN1 (Origene product TP319368, Lot#100814) TP53 (Origene product TP300003, Lot#17J10L19), and GRIA1 (Origene product TP326253, Lot#BJ0A64E).

Techniques: Western Blot, Membrane, Mutagenesis, Control, Two Tailed Test, Fluorescence, Labeling, Staining, Immunofluorescence, Expressing, Purification