Journal: bioRxiv

Article Title: Expansion microscopy at one nanometer resolution

doi: 10.1101/2022.08.03.502284

Figure Lengend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

Article Snippet: The primary antibodies used were anti synaptotagmin1 (SYT1, #105011 Synaptic Systems), anti Homer1 (#160 003, Synpatic Systems), anti Shank2 (#162204 Synaptic Systems), anti GluR2 (Alomone Labs, #AGC-005, Jerusalem, Israel), anti GluN2b (Neuromab 75-101, California, USA), anti MAP2 (Novus Biologicals #NB300-213), anti vGluT1 (#135304, Synaptic Systems), anti Bassoon (#ADI-VAM-PS003-F, Enzo, New York, USA).

Techniques: Labeling, Extraction, MANN-WHITNEY, Immunostaining

Journal: Neuron

Article Title: Regulation of Thalamic and Cortical Network Synchrony by Scn8a

doi: 10.1016/j.neuron.2017.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal mouse anti-GluR2 , Neuromab , Cat# 75-002; RRID: AB_2232661.

Techniques: Plasmid Preparation, Virus, Recombinant, Avidin-Biotin Assay, Software, Imaging

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PP2A bindings to PDI and GluA2 in the hippocampus following KA injection. KA increases PDI:PP2A and GluA2:PP2A bindings in control siRNA-infused animals, but not in PDI siRNA-infused animals. ( a ) Representative Western blot images for the PDI:PP2A and GluA2:PP2A bindings. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PP2A level ( b ) and PDI:PP2A binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test).

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Control, Western Blot, Binding Assay, Saline

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PICK1 bindings to GluA2 in the hippocampus following KA injection. PDI siRNA does not influence PICK1 level in both saline- and KA-treated groups. As compared to control siRNA, PDI siRNA increases GluA2A:PICK1 binding in saline-treated group. Although KA does not affect GluA2:PICK1 binding in control siRNA-infused group, it increases it in PDI siRNA-infused group. ( a ) Representative Western blot images for the GluA2:PICK1 binding. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PICK1 level ( b ) and GluA2:PICK1 binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test). ( d ) Scheme of the role of PDI in AMPAR internalization. PDI may reduce lead to reduction-induced PP2A activation, which would abolish PICK1-mediated AMPAR internalization by dephosphorylating GluA2 S880 and CaMKII T286 sites.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Saline, Control, Binding Assay, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques:

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits.

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: Fig. 4 | Glioma membrane depolarization promotes glioma progression. a, Optogenetic model for glioma depolarization. Blue dots represent ChR2-expressing glioma cells; light blue rectangle denotes region of analysis. P, postnatal day. b, Proliferation index of SU-DIPG- XIII-FL-ChR2 xenografts after mock stimulation or blue light stimulation, measured as percentage of GFP+/HNA+ cells expressing Ki67 (mock stim, n = 8; stim, n = 9 mice). c, As in b but for SU-DIPG-VI-ChR2 xenografts (n = 6 mice per group). d, Representative confocal micrographs from c, illustrating proliferation of SU-DIPG-VI-ChR2 xenografts. Red denotes human nuclei staining by HNA; white denotes Ki67. Scale bar, 50 µm. e, f, Kaplan–Meier survival curves of SU-DIPG-XIII-P* (P denotes pontine tumour) xenografts that overexpress GFP-only (green) or GluA2- WT-GFP (red) (e) and GFP-only (in 80% of cells, green) or GluA2-DN- GFP (in 80% of cells, blue) (f) (n = 5 mice per group). g, Competitive outgrowth of non-GluA2-DN-GFP-expressing cells in f, determined by

Article Snippet: We introduced SpeI (5′-TAAGCAACTAG TATGCAAAAGATTATGCAT-3′) and XmaI (5′-TGCTTACCCGGGC TAAATTTTAACACTTTCGAT-3′) restriction sites in full-length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Membrane, Expressing, Staining

Journal: Journal of neurochemistry

Article Title: Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b

doi: 10.1111/jnc.14231

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: #4027 RRID:AB_1147622 WB (1:1,000) GluR2 P(Tyr 869,873,876) rabbit Cell Signaling Tech.

Techniques: Transduction

Journal: Journal of neurochemistry

Article Title: Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b

doi: 10.1111/jnc.14231

Figure Lengend Snippet: Olfm1 interacts with presynaptic SNARE complex proteins and postsynaptic GluR2 in synaptosomes from adult wt brain. (a) Western blot analysis of indicated proteins in adult wt and olfm1 null zebrafish brain and retina. The blots marked by an asterisk was obtained in the P2 fraction. (b) Quantification of three independent experiments as in A for VAMP and synaptophysin. Data for each protein were normalized using beta-III tubulin level. **p < 0.01. (c) Biochemical fractionation of adult zebrafish brain and intracellular localization of olfm1 and some pre- and post-synaptic proteins in adult zebrafish brain. Note co-purification of olfm1 and several synaptosomal membrane proteins with the LP1 fraction but not with the soluble fraction (S3). (d) Co-immunoprecipitation of Olfm1 with GluR2 from the synaptosomal fraction. The levels of GluR2 and phosphorylated GluR2 were slightly increased in synaptosomes isolated from olfm1 null brain compared with wt brain. (e, f) Co-immunoprecipitation of Olfm1 with VAMP2 and syntaxin1 from brain synaptosomes.

Article Snippet: #4027 RRID:AB_1147622 WB (1:1,000) GluR2 P(Tyr 869,873,876) rabbit Cell Signaling Tech.

Techniques: Western Blot, Fractionation, Copurification, Membrane, Immunoprecipitation, Isolation

Journal: Journal of neurochemistry

Article Title: Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b

doi: 10.1111/jnc.14231

Figure Lengend Snippet: Changes in the localization of GluR2 and VAMP2 in olfm1 null retina and brain. (a) The internalization of GluR2 in olfm1 null and wt larval retinal cells in culture. Retina from 32 hpf larvae were dissected and cultured in vitro for 5 days. External GluR2s were labeled with antibodies against the N-terminal part of GluR2 and allowed to be internalized for 15 min with or without 100 μM AMPA in the culture. The external GluR2 was detected using Alexa488-secondary antibody. The internalized GluR2 was detected using Alexa555-secondary antibody after permeabilization of the cell membrane. Scale bar, 5 μm. (b) The number of green and red puncta was counted and the ratio of the internal and the total labeled GluR2 was calculated. N = 6–8 individual cultures. (c) The levels of GluR2 and phosphorylated GluR2 were slightly increased in synaptosomes isolated from olfm1 null brain compared with wt brain. (d) Reduced palmitoylation of GluR2 precipitated by anti-GluR2 antibody from synaptosomes isolated from olfm1 null brain compared with wt samples (upper panel). Note that olfm1 co-immunoprecipitated with GluR2 was also palmitoylated, indicating that olfm1 may be associated with membranes through its lipid modification as well as its binding to other membrane-associated proteins (lower panel). HAM-untreated precipitates were used as a negative control and they show only weak signals for both GluR2 and Olfm1 (e) The level of VAMP2 was increased only in the synaptosomal LP2 fraction (synaptic vesicles) but not in LP2 (synaptosomal membrane). (f) Quantification of three independent experiments as in (f). *p<0.05,***< 0.001.

Article Snippet: #4027 RRID:AB_1147622 WB (1:1,000) GluR2 P(Tyr 869,873,876) rabbit Cell Signaling Tech.

Techniques: Cell Culture, In Vitro, Labeling, Membrane, Isolation, Immunoprecipitation, Modification, Binding Assay, Negative Control

Journal: Journal of neurochemistry

Article Title: Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b

doi: 10.1111/jnc.14231

Figure Lengend Snippet: Characterization of the microdomain fraction from wt and olfm1 null brain. (a) Changes in the protein levels detected in the microdomain fraction from wt and olfm1 null adult brain. (b) Quantification of the results of three independent experiments as in (a). (c, d) GluR2 localization in CTX-B-labeled lipid rafts in the IPL of adult wt (c) and olfm1 null (d) zebrafish retina. The black/white images in the middle and right rows represent enlarged images in each channel from the red squared areas in the left row. Note that all green circled GluR2-positive puncta are localized in CTX-B bound lipid rafts in wt retina, while some of GluR2 puncta in olfm1 null are not in lipid rafts. Scale bar, 5 μm. (e) Quantification of the results of seven wt and eight olfm1 null independent eye sections as in (c and d). *p<0.05, **p< 0.01, ***p< 0.001.

Article Snippet: #4027 RRID:AB_1147622 WB (1:1,000) GluR2 P(Tyr 869,873,876) rabbit Cell Signaling Tech.

Techniques: Labeling

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Proliferative response of GFP (control) and GluA2-dominant negative subunit expressing glioma cells (GluA2-DN) after 24-hour exposure to soluble extracellular neuroligin-3 (NLGN3; 100nm) in the presence and absence of AMPA-receptor blocker, NBQX (10μM). b , Western blot analysis of phospho-AKT (Ser473) and total AKT in GFP (control) glioma cells in response to 5-minute exposure of soluble extracellular neuroligin-3 (NLGN3; 100nm) in the presence and absence of AMPA-receptor blocker, NBQX (10μM); left. Quantitative analysis of the ratio of pAKT/AKT normalized to vehicle (right). c , Western blot analysis of phospho-AKT (Ser473) and total AKT in GluA2-DN expressing glioma cells in response to 5-minute exposure of soluble extracellular neuroligin-3 (NLGN3; 100nm; left). Quantitative analysis of the ratio of pAKT/AKT normalized to vehicle (right). d , Time course of evoked glioma cell EPSC block by NASPM (100 μM, duration=red bar (n=7/5 cells/mice; left); Representative trace before (black) and after (red) addition of NASPM (right). e, Quantification of (d). f , GluA2 subunit Q/R editing efficiency in SU-DIPGXIII-FL and SU-DIPGVI cells as measured by PCR and expressed as % edited. g , Expression of ADAR1, the enzyme responsible for Q/R editing of GluA2 mRNA. Plot illustrates ADAR1 enzyme mRNA expression relative to beta-actin as measured by qPCR. Analyses in a,b,c,f,g were calculated from three independent sets of cells. Data shown as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s post-hoc analysis (a,b), by two-tailed Student’s t-test (c), by two-tailed paired Student’s t-test (e). All data shown as mean ± s.e.m. *P<0.01, **P<0.001, ***P<0.001, ****P<0.0001, NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Control, Dominant Negative Mutation, Expressing, Western Blot, Blocking Assay, Two Tailed Test

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a, Electrophysiological responses by model. Number of whole cell patch clamp recordings from cells in xenografted hippocampal slices separated by electrophysiological response to local electrical stimulation. b , Demonstration of depolarizing inward current in SU-DIPXIII-FL-ChR2 cells in response to single stimulation and 20Hz pulses of blue light as measured in current clamp (top) and voltage clamp (bottom). c , Proliferation index of xenografted SU-DIPGXIII-FL-YFP control glioma cells (no opsin expressed) in response to blue light stimulation or mock stimulation as measured by the proportion of GFP+/HNA+ cells expressing Ki67 24-hours after five optogenetic stimulation sessions (n=3 mice, mock stim; n=4 mice, stim). d, Quantification of cleaved caspase-3 in xenografted SU-DIPGXIII-FL-YFP control glioma cells in response to blue light stimulation or mock stimulation as measured by total number of HNA+ cells co-labeled with cleaved caspase-3 (n=3 mice/group). e , As in (d), quantification of cleaved caspase-3 in xenografted SU-DIPXIII-FL-ChR2 glioma cells (n=3 mice, mock stim; n=4 mice, stim). f , Validation of GluA2-dominative negative AMPA receptor subunit expressing construct. Representative traces of whole-cell voltage-clamp recording of WT (black) and GluA2-DN expressing (grey) SU-DIPGVI cells in response to 500μM (S)-AMPA (n=6 cells). g , Representative traces of whole-cell voltage-clamp recording in WT (black) and GluA2-DN expressing (grey) SU-DIPGXIII-FL cells in response to 500μM (S)-AMPA (n=6 cells). SU-DIPGXIII-FL cells are unable to homogeneously express the dominant construct, and therefore may be connected to WT GluA2 expressing cells, which accounts for the remaining current in the illustrated trace. Incorporation of the GluA2-DN construct thus results in a significantly abrogated AMPAR-dependent depolarization. Data shown as mean ± s.e.m for (c,d,e). All P-values determined by two-tailed Student’s t-test. NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Patch Clamp, Control, Expressing, Labeling, Biomarker Discovery, Construct, Two Tailed Test

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Optogenetic paradigm for glioma depolarization. ChR2-expressing glioma (blue), region of analysis (light blue). b , Proliferation index of SU-DIPGXIII-FL-ChR2 xenograft after mock stimulation (mock stim) or blue light stimulation (stim) measured as percent of GFP+/HNA+ cells expressing Ki67 (mock stim, n=8; stim, n=9 mice). c , As in (b), but SU-DIPGVI-ChR2 xenografts (n=6 mice/group). d , Representative confocal micrographs from (c), illustrating proliferating SU-DIPGVI-ChR2. Red=human nuclei; white=Ki67. Scale bar=50μm. e-f , Kaplan-Meier survival curves of SU-DIPGXIII-P* xenografts overexpressing e, GFP-only (green) or GluA2-WT-GFP (red) and f, GFP-only (in 80% of cells, green) or GluA2-DN-GFP (in 80% of cells, blue); n=5 mice/group. g , Competitive outgrowth of non-GluA2-DN-GFP-expressing cells in (f), determined by GFP/total human nuclei pixel intensity; (n=3 mice/group). h , Representative confocal micrographs of (f-g). White=human nuclei; green=GFP. Scale bar=50μm. i , Representative confocal images of SU-DIPGXIII-FL xenografts expressing GFP-only control (top) or GluA2-DN-GFP (bottom). Gray=MBP; White=glioma-GFP. Scale bar=500μm. j , Quantification of (i) (n=8 mice/group). k , Proliferation index of SU-DIPGVI xenografts treated with perampanel (AMPAR blocker) or vehicle control; (n=8 mice/group). l , Proliferation index of SU-DIPGXIII-FL in mice treated with meclofenamate (gap junction blocker) or vehicle control; (n=9 vehicle, n=8 treated mice). Data shown as mean±s.e.m (b,c,g,j,k,l). **P<0.01. ***P<0.001, ****P<0.0001. P-values determined by two-tailed unpaired Student’s t-test (b,c,g,k,l); two-tailed log rank analyses (e,f); two-sided Mann-Whitney test (j).

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Expressing, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Kaplan-Meier survival curves of second cohort of mice orthotopically xenografted with control GFP-only or GluA2-DN-GFP over-expressing cells (SU-DIPGXIII-P* xenograft model; n=5 mice per group). b , Representative coronal sections of mouse brains bearing SU-DIPGXIII-FL xenografts either expressing control GFP construct (left) or GluA2-DN-GFP construct; right). Gray, MBP; White, glioma-GFP. c , Proliferation indices of SU-DIPGXIII-FL cells at baseline in neuronal medium, in response to 10μM NBQX, in co-culture with neurons, or in co-culture with neurons in the presence of 10μM NBQX (n=3 biological replicates/group, except n=4 for baseline). d, Representative images of neuron-glioma co-cultures in the presence and absence of NBQX. Green = neurofilament (neuronal processes); Red = nestin (glioma cell processes); White = Ki67. Scale bar = 50μm. e , in vitro growth analysis of control GFP or GluA2-DN-GFP cells monitored over 3 days. f, in vitro apoptosis analysis of control GFP or GluA2-DN-GFP as measured by % of total cells co-stained with cleaved-caspase. g , 3D Matrigel invasion assay in WT (GFP) and GluA2-DN (GluA2-DN-GFP) expressing SU-DIPGXIII-FL cells 72 hours after seeding. h , Representative images of (g) at time 0 hr (left) and 72 hr (right) in control GFP-expressing (top) and GluA2-DN-GFP expressing cells (bottom). Scale bar = 1000μm. i , 3D migration assay in WT (GFP) and GluA2-DN (GluA2-DN-GFP) expressing SU-DIPGXIII-FL cells 72 hours after seeding. j , Representative images of (i) at time 0 hr (left) and 72 hr (right) in control GFP-expressing (top) and GluA2-DN-GFP expressing cells (bottom). Scale bar =1000μm. k, Representative confocal micrographs illustrating proliferating SU-DIPGVI cells in vehicle or perampanel-treated mice (n=8 mice/group). Red = human nuclei; white = Ki67. Scale bar = 50μm. l , IVIS bioluminescence analysis of overall tumor growth in SU-DIPGXIII-FL xenografts treated with vehicle or meclofenamate over a two-week period. Data represented as fold change in total flux; n=5 mice/group. Data shown as mean ± s.e.m. for (c,e,f,g,i,l). For analyses in (d-j), n=3 biological replicates. P-values determined by two-tailed log rank analyses (a), by one-way ANOVA with post-hoc analysis (c), by two-tailed unpaired Student’s t-test (f,g,i,l). *P<0.05, **P< 0.01,****P<0.0001. NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Control, Expressing, Construct, Co-Culture Assay, In Vitro, Staining, Invasion Assay, Migration, Two Tailed Test