anti glun2b  (Cell Signaling Technology Inc)

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Expression of GluN2A (A) and GluN2B (B) and basal extracellular levels of L‐glutamate (C) and D‐serine (D) in 4‐weeks and 8‐weeks of age S286L‐TG and wild‐type littermate. Ordinates indicate mean ± SD ( n = 6) of (A) expression levels of GluN2A relative to GAPDH in the plasma membrane fraction (B) expression levels of GluN2B relative to GAPDH in the plasma membrane fraction, (C) basal extracellular L‐glutamate level (μM) and (D) basal extracellular D‐serine level (μM) in the frontal cortex of wild‐type (gray column) and S286L‐TG (blue column). The lower‐side panels in A and B indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to 4‐weeks of age (4 W) and # p < 0.05 relative to wild‐type using two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (A) expression of GluN2A ( F age [1, 20] = 46.7 [ p < 0.05], F genotype [1, 20] = 5.34 [ p < 0.05], F age*genotype [1, 20] = 1.1 [ p > 0.05]), (B) expression of GluN2B ( F age [1, 20] = 22.4 [ p < 0.05], F genotype [1, 20] = 8.3 [ p < 0.05], F age*genotype [1, 20] = 2.0 [ p > 0.05]), (C) L‐glutamate level ( F age [1, 20] = 3.2 [ p > 0.05], F genotype [1, 20] = 21.2 [ p < 0.05], F age*genotype [1, 20] = 1.9 [ p > 0.05]) and (D) D‐serine level ( F age [1, 20] = 8.4 [ p < 0.05], F genotype [1, 20] = 21.6 [ p < 0.05], F age*genotype [1, 20] = 2.8 [ p > 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Clinical Proteomics, Membrane, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic administration of probenecid, MK‐801, memantine, and FP802 on expression of GluN2A and GluN2B in S286L‐TG and wild‐type littermates. All rats were chronically administered by vehicle (control), probenecid (PBN: 100 mg/kg/day), MK‐801 (0.1 mg/kg/day), memantine (MEM: 10 mg/kg/day) and FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of expression levels of GluN2A (A1‐A4) and GluN2B (B1‐B4) relative to GAPDH in wild‐type (A1‐A2, B1‐B2) and S286L‐TG (A3‐A4, B3‐B4). The right‐side panels indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control using one‐way ANOVA with Scheffe's post hoc test. F ‐values regarding effects of probenecid and MK‐801 on GluN2A expression in wild‐type (A1) ( F [2, 15] = 7.8 [ p < 0.05]), GluN2A in S286L (A3) ( F [2, 15] = 19.4 [ p < 0.05]), GluN2B in wild‐type (B1) ( F [2, 15] = 12.1 [ p < 0.05]) and GluN2B in S286L‐TG (B3) ( F [2, 15] = 18.2 [ p < 0.05]). F ‐values regarding effects of memantine and FP802 on GluN2A in wild‐type (A2) ( F [2, 15] = 0.4 [ p > 0.05]), GluN2A in S286L‐TG (A4) ( F [2, 15] = 7.1 [ p < 0.05]), GluN2B in wild‐type (B2) ( F [2, 15] = 0.2 [ p > 0.05]) and GluN2B in S286L‐TG (B4) ( F [2, 15] = 4.8 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic combined administration of memantine with FP802 on ADSHE seizure frequency (A), sucrose preference (B), expression of GluN2A (C1) and GluN2B (C2), and basal extracellular levels of L‐glutamate (D) and D‐serine (E) in S286L‐TG and wild‐type littermate. All rats were chronically administered by vehicle (control) and combined of memantine (MEM: 10 mg/kg/day) with FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of (A) ADSHE seizure frequency (count h −1 ), (B) consumption of sucrose preference (%), (C1) expression levels of GluN2A relative to GAPDH, (C2) expression levels of GluN2B relative to GAPDH, (D) basal extracellular L‐glutamate level (μM) and (E) basal extracellular D‐serine level (μM). The right‐side panels in C1‐C2 indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control and # p < 0.05 relative to wild‐type using student T ‐test or one‐way or two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (B) sucrose preference: MEM ( F memantine+FP802 [1, 20] = 21.3 [ p < 0.05], F genotype [1, 20] = 5.1 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 5.3 [ p < 0.05]), (D) L‐glutamate level: ( F memantine+FP802 [1, 20] = 5.3 [ p < 0.05], F genotype [1, 20] = 15.9 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 4.8 [ p < 0.05]), (E) D‐serine level: ( F memantine+FP802 [1, 20] = 7.6 [ p < 0.05], F genotype [1, 20] = 22.4 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 10.4 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a , b , The representative immunoblots ( a ) and quantitative analyses ( b ) of Glun1, Glun2A, Glun2B and Syn1 in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 4 per group. c , RT–qPCR assays mRNA expression of the Glun1, Glun2A, Glun2B and Syn1 in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 5 per group. d , ChIP–qPCR analysis of the enrichment of H3K9bhb at Glun1, Glun2A, Glun2B, Glun2C and Syn1 promoters in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 5 per group. e , f , Supplementing with β-OHB could increase the density of 3xTg-AD dendritic spines detected by Golgi-cox staining; the representative images ( e ) and quantitative analysis ( f ) of spine, n = 5 per group, three fields per mice. Scale bar, 5 μm. g – j , The Sholl analysis showed the synaptic complexity of neurons after supplementing with β-OHB in 3xTg-AD mice; the representative images ( g and i ) and the quantitative analysis ( h and j ), n = 5 per group, two fields per mice. Scale bar, 50 μm. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d and f . Two-way ANOVA followed by Bonferroni’s post hoc test for i and k . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2A , NMDAR2A , Poly- , 1:1000 , Proteintech , 28525-1-AP.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, ChIP-qPCR, Staining

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a – c , The HMGCS2 upregulation promotes the protein ( a and b ) and mRNA ( c ) expression of H3K9bhb, Glun1, Glun2A, Glun2B, Syn1 and PSD95, n = 4 or 5 per group. d , e , ChIP–qPCR analyses of the enrichment of H3K9bhb at Glun1, Glun2A, Glun2B and Syn1 promoters in the primary neurons of the WT, 3xTg-AD and 3xTg-AD + HMGCS2 mice n = 5 per group ( d ) and representative gel images from ChIP–qPCR assays ( e ). f – j , The HMGCS2 upregulation promotes the expression of Syn1 (scale bar, 25 μm) ( f ); n = 10 cells per group in MAP2 immunofluorescence ( g ) and quantitative analysis ( h ), n = 10 cells per group and SYP ( i and j ), n = 10 cells per group, scale bar, 15 μm. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d and j . Two-way ANOVA followed by Bonferroni’s post hoc test for h . * P < 0.05, ** P < 0.01 , *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2A , NMDAR2A , Poly- , 1:1000 , Proteintech , 28525-1-AP.

Techniques: Expressing, ChIP-qPCR, Immunofluorescence

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a , b , A western blot analysis ( a ) of hippocampal lysates shows that HMGCS2 upregulation increases the protein levels ( b ) of H3K9bhb, Glun1, Glun2A, Glun2B, Syn1 and PSD95, n = 3 per group. c , The ChIP–qPCR analysis of H3K9bhb enrichment at the promoters of Glun2A , Glun2B , Syn1 and PSD95 in the four groups, n = 5 per group. d , The mRNA levels of Glun1, Glun2A, Glun2B, Syn1 and PSD95 in the hippocampus, as determined by RT–qPCR, n = 5 per group. e , f , Golgi staining reveals increased dendritic spine density in 3xTg-AD mice following overexpression of HMGCS2; representative images ( e ) and quantification ( f ) are shown, n = 5 per group, three fields per mice. Scale bar, 5 μm. g – j , A behavioral assessment of spatial learning and memory using the MWM, NOR and contextual fear conditioning tests: area under the curve (AUC) of escape latency during MWM training of day 1–6 ( g ), escape latency on day 7 of the MWM test ( h ), NOR discrimination index ( i ), freezing time on day 7 in the contextual fear conditioning test ( j ), n = 8 per group. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d , f and g – j . * P < 0.05, ** P < 0.01 , *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2A , NMDAR2A , Poly- , 1:1000 , Proteintech , 28525-1-AP.

Techniques: Western Blot, ChIP-qPCR, Quantitative RT-PCR, Staining, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP1 and ZIP3 localization in the DCN. A . Immunofluorescent imaging of DCN slices labeled with ZnT3 (red) and the postsynaptic markers GluN2A or CaMKII (green). B . Immunofluorescent staining of ZnT3, ZIP1 or ZIP3 (red) with the synaptic terminal marker VGLUT1 (green). C . GluN2A or CaMKII (green) immunofluorescent co-labeling with ZnT1, ZIP1 or ZIP3 (red), bottom panels show colocalization analyses, represented by Mander’s colocalization coefficient, calculated between the postsynaptic marker and each of the Zn 2+ transporters. For GluN2A colocalization, Tukey’s multiple comparisons test analysis shows a significant difference between ZnT3 and ZIP3 p-value: 0.016, as well as between ZnT3 and ZnT1 p-value: 0.031, F (1.669, 4.451) = 22.32. In case of CaMKII, Tukey’s multiple comparisons test shows significance for ZIP3 p-value: 0.009 and ZnT1 p-value: 0.016, F (3, 15) = 7.202. D . Immunofluorescent staining of PAX6 or Tbr2 (green) with ZIP1 or ZIP3 (red). The colocalization analyses for PAX6, bottom panel, shows Mander’s coefficients of the two Zn 2+ transporters (Mann Whitney test, p-value: 0.003). White arrows indicate examples of cells that show co-localization of the markers

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Imaging, Labeling, Staining, Marker, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP3 and ZnT1 are in direct contact, which enhances ZnT1 efflux rates. A. Co-immunoprecipitation assay in SH-SY5Y cells over expressing ZnT1 and ZIP3, with or without the murine subunit GluN2A (mGluN2A) or the human glutamate receptor (hGluN2A) subunit. The samples were immunoprecipitated with an antibody against ZnT1. The lysates were then separated on SDS-PAGE and subjected to immunoblotting with antibodies against ZIP3, representative of 3 repetitions. Lysis buffer shown as control. B. Co-immunoprecipitation in HEK293 cells expressing ZnT1 and ZIP3. Samples were immunoprecipitated either with an antibody against ZnT1 (left) or ZIP3 (right). Lysates were then separated on SDS-PAGE and subjected to immunoblotting with antibodies against ZIP3 or ZnT1, representative of 3 repetitions. Lysisbuffer shown as control. Note that expected MW for ZIP3 is 37kDa, identified MW likelyrepresents its dimerization and glycosylation; full blots shown in Supplementary Information. C. Representative traces of FluoZin-3 fluorescence changes in SH-SY5Y cells expressing ZnT1+ZIP3 (orange) or ZnT1+PCDNA as control (blue). Cells were perfused with 200μM Zn2+ in Ringer’s solution added with or without pyrithione (see Methods) at the indicated time. The initial Zn 2+ influx and efflux rates were monitored and compared between the two conditions. Unpaired ttest analysis, *** p-value: 0.0003, t(28)=4.131

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, SDS Page, Western Blot, Lysis, Control, Glycoproteomics, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP3- ZnT1 physical interaction in DCN cartwheel cells. A . Proximity ligation assay (PLA) performed on DCN slices using probes for GluN2A and either ZnT1, ZIP3, or ZIP1. Analysis of PLA puncta (red) was done on the molecular layer (outlined in white) to assess protein–protein interactions in postsynaptic cells of the parallel fibers. The graph (right panel) quantifies PLA puncta normalized to ZnT1-GluN2A puncta. Statistical analysis Dunnett’s multiple comparisons test, p-value: 0.0014, F (2, 11) = 11. B . Co-immunoprecipitation assay using DCN and hippocampal lysates. Protein samples were immunoprecipitated with antibodies against GluN2A, followed by SDS-PAGE separation and immunoblotting with antibodies against ZIP3 to confirm physical interactions. Note that expected MW for ZIP3 is 37 kDa, identified MW likely represents its dimerization and glycosylation; full blots shown in Supplementary Information. C . Schematic representation of Zn 2+ transporters in DCN, indicating the ZIP3-ZnT1-GluN2A complex on the cartwheel cells of the DCN

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Proximity Ligation Assay, Protein-Protein interactions, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Western Blot, Glycoproteomics

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP1- ZnT1 physical interaction in the hippocampus. A . Co-immunoprecipitation assay using SH-SY5Y cells that were immunoprecipitated with ZnT1 and then exposed to ZIP1 (left panel) or ZIP3 (right panel) antibodies. Note that the right panel is taken from the gel presented in Fig. , the right lane for mGluN2A-ZIP3-ZnT1 expressing cells overlaps with that figure. Note that expected MW for ZIP3 is 37 kDa, identified MW likely represents its dimerization and glycosylation; full blots shown in Supplementary Information. B . Representative traces of fluorescent imaging of FluoZin-3 changes in SH-SY5Y cells expressing ZnT1 + ZIP1 (green) or ZnT1 + PCDNA as control (blue). Cells were perfused with 200 µM Zn 2+ in Ringer’s solution added at the indicated time, with or without pyrithione (see methods). The initial Zn 2+ influx (middle panel) and efflux (right panel) rates are shown in the bar graphs. Unpaired t-test analysis, *** p-value: 0.0001, t(23) = 5.613. C . Co-immunoprecipitation assay using DCN and hippocampal lysates. Protein samples were immunoprecipitated with antibodies against ZIP1, followed by SDS-PAGE separation and immunoblotting with antibodies against GluN2A. D . Proximity ligation assay (PLA) performed on CA3 hippocampal slices using probes for GluN2A and either ZnT1 or ZIP1. Red puncta represent the GluN2A-ZnT1 or ZIP1 interaction in the CA3 pyramidal cell layer (marked by white lines). Analysis of PLA puncta, performed with the GluN2A and either ZnT1, ZIP1 or ZIP3, on CA3 pyramidal layer to assess protein–protein interactions in postsynaptic cells. The graph (bottom panel) quantifies PLA puncta normalized to ZnT1-GluN2A puncta. Statistical analysis Dunnett’s multiple comparisons test, p-value: 0.013, F (2, 6) = 9.81. E . Schematic presentation of the Zn 2+ -cycle proteins, ZIP1-ZnT1-GluN2A, expressed on the postsynaptic CA3 pyramidal cells that are adjacent to the ZnT-3 and ZIP3 expressing mossy fiber terminals, consistent with previous work

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing, Glycoproteomics, Imaging, Control, SDS Page, Western Blot, Proximity Ligation Assay, Protein-Protein interactions