rabbit polyclonal anti nr2a  (Cell Signaling Technology Inc)

Journal: Frontiers in Cellular Neuroscience

Article Title: Rescue of sharp wave-ripples and prevention of network hyperexcitability in the ventral but not the dorsal hippocampus of a rat model of fragile X syndrome

doi: 10.3389/fncel.2023.1296235

Figure Lengend Snippet: Western blotting analysis of the three NMDAR subunits, NR1, NR2A, and NR2B in WT and KO dorsal (A) and ventral hippocampus (B) . The results of the statistical analysis (independent t -test) showed similar expressions between the WT and KO dorsal hippocampus (NR1: t 14 = 0.107, p = 0.813; NR2A: t 14 = –0.24, p = 0.369; NR2B: t 14 = 1.128, p = 0.842; n = 8 rats in either genotype), and ventral hippocampus (NR1: t 14 = –0.241, p = 0.813; NR2A: t 14 = 0.803, p = 0.435; NR2B: t 14 = 0.203, p = 0.842; n = 8 rats in either genotype). Also, the NR2A/NR2B ratio is similar in WT and KO dorsal ( t 10.15 = –1.38, p = 0.199) and ventral hippocampus ( t 9.72 = 0.383, p = 0.71). The expression of NR2A subunit and the NR2A/NR2B ratio is higher in the dorsal compared with the ventral hippocampus in both WT (NR2A: t 14 = 4.27, p < 0.001, and NR2A/NR2B ratio: t 14 = 3.08, p = 0.008) and KO rats (NR2A: t 14 = 4.017, p = 0.001, and NR2A/NR2B ratio: t 14 = 3.01, p = 0.009). In contrast, NR1 and NR2B are similarly expressed in WT and KO dorsal (NR1: t 14 = 1.308, p = 0.212, and NR2B: t 14 = 1.09, p = 0.294) and ventral hippocampus (NR1: t 14 = 1.039, p = 0.316, and NR2B: t 14 = 0.187, p = 0.854). (C) Images of individual western blot samples with detected bands of the NMDA receptor protein subunits, and the corresponding loading marker band of beta actin. “ns” denotes not significant difference.

Article Snippet: Membranes were next incubated overnight at 4°C with the following primary antibodies diluted in 3% PBST: rabbit anti-α1 GABA A R polyclonal antibody (1:2500 #06-868, Millipore Sigma), rabbit anti-NR1 monoclonal antibody (1:1000 #D65B7, Cell Signaling), rabbit anti-NR2A polyclonal antibody (1:1000 #4205, Cell Signaling), rabbit anti-NR2B monoclonal antibody (1:1000 #B8E10, Cell Signaling) and rabbit anti-β-actin polyclonal antibody (1:15000 #E-AB-20058, Elabscience).

Techniques: Western Blot, Expressing, Marker

Journal: bioRxiv

Article Title: Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

doi: 10.1101/2023.04.01.535233

Figure Lengend Snippet: The alternations as a result of variants in NR2A subunit on the expression and function of trafficking-deficient NMDARs. a , Upper panel, cartoon representation of tetrameric NMDARs, with the NR1 subunits in light and dark grey and the NR2A subunits in green and blue (PDB: 7EOS ), each subunit consisting of an extracellular amino-terminal domain (ATD), an extracellular ligand-binding domain (LBD), a transmembrane domain comprised of three transmembrane helices and one reentrant loop (TMD), and an intracellular carboxy-terminal domain (CTD). The positions of the M705V and A727T are highlighted as spheres in LBD. Lower panel, linear representation of an NR2A subunit. The LBD contains two segments of S1 and S2. b , The NR2A expression level in transiently transfected HEK293T cells compared to the cortex and hippocampus tissue in mouse brain. NS:not significant. c , The effect of M705V and A727T on the total protein expression level of NR2A. β-actin serves as the loading control of total protein lysates. d , The effect of M705V and A727T on the surface protein expression level of NR2A as determined by surface biotinylation assays. Na + /K + -ATPase serves as the loading control of membrane proteins. e , Surface NR2A staining was in green (column 1), and plasma membrane marker Na + /K + -ATPase staining was in red (column 2). Merge of these two signals with the nucleus stained in blue with DAPI was shown in column 3. Scale bar = 20 μm. The fluorescence intensity of the surface NR2A was quantified from 30-50 cells per condition as shown on the lower panel. f , Automated patch-clamping was performed with the IonFlux Mercury 16 ensemble recording at a holding potential of -60 mV. Glutamate (10 mM) and glycine (100 µM) were applied simultaneously for 3 s, as indicated by the horizontal bar above the currents. The peak currents (Imax) were acquired and analyzed by the Fluxion Data Analyzer (n = 9 - 10 ensemble recording; each ensemble recording enclosed 20 cells). g , Inhibition of proteasomal degradation (MG132, 10 µM, 6 h) and lysosomal degradation (BafA1, 1 µM, 6 h) independently demonstrated that NR2A subunits containing either M705V or A727T variants are mainly degraded by the proteasome pathway. Each data point is reported as mean ± SEM. One-way ANOVA followed by a post-hoc Tukey test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The rabbit polyclonal anti-NR2A (catalog #: ab124913; 1:3,000), mouse polyclonal anti- NR2A (catalog #: ab240884; 1:100), and rabbit monoclonal anti-Na + /K + -ATPase antibody (catalog #: ab76020; 1:3,000) were purchased from Abcam.

Techniques: Expressing, Ligand Binding Assay, Transfection, Staining, Marker, Fluorescence, Inhibition

Journal: bioRxiv

Article Title: Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

doi: 10.1101/2023.04.01.535233

Figure Lengend Snippet: Effects of BIX on the expression and function of trafficking-deficient variants in NR2A subunit of NMDARs. a , Dose-response analysis of BIX treatment (24 h) on the total protein levels of NR2A subunits in HEK293T cells expressing NR1NR2A(M705V) or NR1NR2A(A727T). b , The toxicity of BIX treatment (24h) was quantified with a resazurin assay to determine cell viability under different concentrations of BIX application. Tg (2 µM, 6 h) was used as a positive control. Tg: thapsigargin. c , Time-course analysis of BIX (10 μM) treatment on the total protein levels of NR2A subunits in HEK293T cells expressing NR1NR2A(M705V) or NR1NR2A(A727T). β-actin serves as the total protein loading control. d, BIX treatment (10 μM, 24 h) increases surface expression of variant NMDARs. Surface NR2A staining was in green (column 1), and plasma membrane marker Na + /K + -ATPase staining was in red (column 2). Merge of these two signals with the nucleus stained in blue with DAPI was shown in column 3. Scale bar = 20 μm. The fluorescence intensity of the surface NR2A was quantified from 30-50 cells per condition. e , Surface biotinylation assays further demonstrated that BIX (10 µM, 24 h) enhances surface expression of variant NMDARs. Na + /K + -ATPase serves as the loading control of membrane protein. f , BIX (10 μM, 24 h) restores function of variant NMDARs as ion channels, as shown by whole-cell patch-clamping recordings. Glutamate (10 mM) and glycine (100 µM) were applied simultaneously for 3 s, as indicated by the horizontal bar above the currents. The peak currents (Imax) were acquired and analyzed by the Fluxion Data Analyzer (n = 7 - 9 ensemble recording; each ensemble recording enclosed 20 cells). Student’s t-test (for comparison of two groups) or one-way ANOVA followed by a post- hoc Tukey test (for comparison of three or more groups) was used for statistical analysis. Each data point is presented as mean ± SEM *, p< 0.05; **, p< 0.01, ***, p < 0.001.

Article Snippet: The rabbit polyclonal anti-NR2A (catalog #: ab124913; 1:3,000), mouse polyclonal anti- NR2A (catalog #: ab240884; 1:100), and rabbit monoclonal anti-Na + /K + -ATPase antibody (catalog #: ab76020; 1:3,000) were purchased from Abcam.

Techniques: Expressing, Resazurin Assay, Positive Control, Variant Assay, Staining, Marker, Fluorescence

Journal: bioRxiv

Article Title: Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

doi: 10.1101/2023.04.01.535233

Figure Lengend Snippet: Effect of BIX treatment on the folding, trafficking, and degradation of variant NMDARs. a , Effect of BIX (10 μM, 24 h) on the folding of NR2A subunits in HEK293T cells stably expressing NR1NR2A(M705V). The normalized ratio of the soluble to insoluble NR2A was shown on the bottom panel. b , BIX (10 μM, 24 h) increases the Endo H-resistant post-ER glycoform of the NR2A subunit in HEK293T cells stably expressing NR1NR2A(M705V). PNGase F, which cleaves all glycans in a glycoprotein, served as a non-glycosylated form of NR2A. Quantification of the Endo H resistant/total NR2A band intensities was shown on the bottom panel. c , Effect of BIX (10 μM, 24 h) on the degradation of the NR2A subunit in HEK293T cells stably expressing NR1NR2A(M705V) using cycloheximide (CHX)-chase analysis. Each data point was reported as mean ± SEM. Student’s t-test (for comparison of two groups) or two-way ANOVA followed by a post-hoc Tukey test (for comparison of three or more groups) was used for statistical analysis. *, p < 0.05; **, p < 0.01.

Article Snippet: The rabbit polyclonal anti-NR2A (catalog #: ab124913; 1:3,000), mouse polyclonal anti- NR2A (catalog #: ab240884; 1:100), and rabbit monoclonal anti-Na + /K + -ATPase antibody (catalog #: ab76020; 1:3,000) were purchased from Abcam.

Techniques: Variant Assay, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

doi: 10.1101/2023.04.01.535233

Figure Lengend Snippet: Effect of BIX on increasing the NR2A variant total protein level is not dependent on BiP. a , Effect of BIX (10 μM, 24 h) on the ER proteostasis network components, including BiP, CRT, CANX, and GRP94, in HEK293T cells stably expressing NR1NR2A(M705V). CRT: calreticulin. CANX: calnexin. Quantifications of the normalized band intensities were shown on the lower panels. b , Effect of BiP inhibition on BIX’s effect to increase the total expression levels of NR2A and BiP in HEK293T cells stably expressing NR1NR2A(M705V) according to western blot analysis. SubAB (0.5 μg/mL, 6 h), a potent BiP-specific protease, was applied to cell culture media to deplete BiP. SubAA272B served as a negative control of SubAB. Tg (thapsigargin) served as a positive control to increase the BiP protein level. c , Effect of BiP overexpression on the total protein levels of NR2A in HEK293T cells stably expressing NR1NR2A(M705V). Each data point is reported as mean ± SEM. Student’s t-test (for comparison of two groups) or one-way ANOVA followed by a post-hoc Tukey test (for comparison of three or more groups) was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The rabbit polyclonal anti-NR2A (catalog #: ab124913; 1:3,000), mouse polyclonal anti- NR2A (catalog #: ab240884; 1:100), and rabbit monoclonal anti-Na + /K + -ATPase antibody (catalog #: ab76020; 1:3,000) were purchased from Abcam.

Techniques: Variant Assay, Stable Transfection, Expressing, Inhibition, Western Blot, Cell Culture, Negative Control, Positive Control, Over Expression

Journal: bioRxiv

Article Title: Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

doi: 10.1101/2023.04.01.535233

Figure Lengend Snippet: BIX increases variant NR2A subunits expression through pharmacological activation of the unfolded protein response (UPR). a , Effect of BIX (10 μM, 24 h) on the total protein levels of NR2A and UPR-associated proteins, including ATF6-N, XBP-1s and CHOP, in HEK293T cells stably expressing NR1NR2A(M705V). Tg (thapsigargin) serves as a positive control to activate the UPR. b , and c , Effect of ATF6-N ( b ) and XBP-1s ( c ) overexpression on the total protein levels of NR2A in HEK293T cells stably expressing NR1NR2A(M705V). d , XPB-1s overexpression enhances surface protein levels of NR2A in HEK293T cells stably expressing NR1NR2A(M705V). Na + /K + -ATPase serves as the loading control of surface protein. e, Effect of an IRE1 inhibitor 4µ8C (32 µM, 24h) on the BIX (10 µM, 24h)-induced protein levels of NR2A in HEK293T cells stably expressing NR1NR2A(M705V). Each data point is reported as mean ± SEM. Student’s t-test (for comparison of two groups) or one-way ANOVA followed by a post-hoc Tukey test (for comparison of three or more groups) was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The rabbit polyclonal anti-NR2A (catalog #: ab124913; 1:3,000), mouse polyclonal anti- NR2A (catalog #: ab240884; 1:100), and rabbit monoclonal anti-Na + /K + -ATPase antibody (catalog #: ab76020; 1:3,000) were purchased from Abcam.

Techniques: Variant Assay, Expressing, Activation Assay, Stable Transfection, Positive Control, Over Expression

Journal: bioRxiv

Article Title: Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

doi: 10.1101/2023.04.01.535233

Figure Lengend Snippet: Our working model presents BIX’s capability to enhance the proteostasis network of NMDARs harboring NR2A mutations by activation of the IRE1 pathway to enhance ER folding capacity and to attenuate variant NR2A degradation through ERAD. Activation of the IRE1 arm of the UPR results in the splicing of XBP1, which in turn acts as a transcription factor to upregulate genes associated with protein folding, assembly, trafficking, and degradation . Together, enhanced folding and assembly and reduction of ERAD of variant NR2A subunits result in an increased population of mature NMDARs that can traffic through the Golgi and to the plasma membrane to perform their function as ion channels. Asterisk (*) represents mutations on the NR2A subunits.

Article Snippet: The rabbit polyclonal anti-NR2A (catalog #: ab124913; 1:3,000), mouse polyclonal anti- NR2A (catalog #: ab240884; 1:100), and rabbit monoclonal anti-Na + /K + -ATPase antibody (catalog #: ab76020; 1:3,000) were purchased from Abcam.

Techniques: Activation Assay, Variant Assay

Journal: BMC Neuroscience

Article Title: Expression of AMPA and NMDA receptor subunits in the cervical spinal cord of wobbler mice

doi: 10.1186/1471-2202-7-71

Figure Lengend Snippet: ionotropic glutamate receptors in the lamina IX in the cervical spinal spinal cord of four-week-old wobbler mice . UPPER PANELS. Representative photomicrographs showing the patterns of GFAP immunostaining in the cervical spinal cord region of 12-week-old mice (A) and age-matched symptomatic wobbler mice (B). CENTRAL PANELS (C-V). Triple staining experiments using Nissl (C, G, K, O, S, purple colour) GFAP (D, H, L, P, T, red colour) and GluR 1–4 or NR2A (E, I, M, Q, U, green colour) in the anterior horn of four-week-old wobbler mice. The merge between the three different colours is shown in panels F, J, N, R, V. LOWER PANELS (W-Z). Triple staining showing Nissl (W), GFAP (X), GluR4 (Y) and the merge between the three different colurs (Z), in the white matter in the anterior region of the cervical spinal cord, four-week-old wobbler mouse. Scale bar, A-B, 100 μm. C-V, 30 μm. W-Z 40 μm.

Article Snippet: For the NMDA receptor we used an anti-mouse monoclonal NR1 antibody (Catatolg No 32-0500, Zymed, Invitrogen, Carlsbad, CA, US; 1:100) and an anti-rabbit polyclonal NR2A antibody (Molecular Probes, Invitrogen, Carlsbad, CA, US; 1:100).

Techniques: Immunostaining, Staining

Journal: BMC Neuroscience

Article Title: Expression of AMPA and NMDA receptor subunits in the cervical spinal cord of wobbler mice

doi: 10.1186/1471-2202-7-71

Figure Lengend Snippet: NMDA receptor subunits in spinal cord of wobbler mice . Representative photomicrographs showing NR1 (A,C) and NR2A (B,D) immunostaining in the cervical spinal cord of four-week-old wobbler mice (B). Scale bar, A, B 100 μm. C, D 20 μm.

Article Snippet: For the NMDA receptor we used an anti-mouse monoclonal NR1 antibody (Catatolg No 32-0500, Zymed, Invitrogen, Carlsbad, CA, US; 1:100) and an anti-rabbit polyclonal NR2A antibody (Molecular Probes, Invitrogen, Carlsbad, CA, US; 1:100).

Techniques: Immunostaining

Journal: BMC Neuroscience

Article Title: Expression of AMPA and NMDA receptor subunits in the cervical spinal cord of wobbler mice

doi: 10.1186/1471-2202-7-71

Figure Lengend Snippet: western blotting analysis and quantification of the percentage of AMPA and NMDA receptor subunits expression in spinal cord of wobbler mice . Representative immunoblot from cervical spinal cord samples from 12-week-old healthy mice and age-matched wobbler mice. Left column: whole spinal cord homogenates. Right column: spinal cord TIF. Quantification of the mean values of immunodensity for CaMK, AMPA and NMDA receptor subunits, obtained from the cervical spinal wobbler mice in whole spinal cord homogenates (Homo) and in TIF. Data are representative of two independent experiments on two different homogenate preparations of four pooled animals for each group and replicated two times in each homogenate preparation. Values reported represent the percentage of the mean values of immunodensity obtained in 12-week-old wobbler mice compared to the levels measured in healthy littermates and normalized to 100. Both preparations samples were analyzed by Western blot analysis with CaMKII, GluR1, GluR2, GluR3, GluR4, NR1, and NR2A antibodies. The same amount of protein was loaded per lane.

Article Snippet: For the NMDA receptor we used an anti-mouse monoclonal NR1 antibody (Catatolg No 32-0500, Zymed, Invitrogen, Carlsbad, CA, US; 1:100) and an anti-rabbit polyclonal NR2A antibody (Molecular Probes, Invitrogen, Carlsbad, CA, US; 1:100).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Brimonidine Blocks Glutamate Excitotoxicity-Induced Oxidative Stress and Preserves Mitochondrial Transcription Factor A in Ischemic Retinal Injury

doi: 10.1371/journal.pone.0047098

Figure Lengend Snippet: BMD or vehicle were injected once by IP at 24 hours before and at the time of initial IOP elevation for 24 hours after ischemia. (A) Vehicle-treated ischemic retina significantly increased GFAP protein expression compared with control retina. In contrast, BMD significantly decreased GFAP protein expression in ischemic retina. (B–E) GFAP immunohistochemisty. When the primary antibody for GFAP was omitted, there was no labeling of the secondary antibody (B). In comparison with control retina (C), vehicle-treated ischemic retina showed activation of müller cells (arrowheads) and astrocytes (D). In contrast, BMD significantly decreased activation of müller cells and astrocytes (E). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale = 20 µm. (F) Vehicle-treated ischemic retina significantly increased NR1, NR2A and SOD2 protein expression compared with control retina. In contrast, BMD significantly decreased NR1, NR2A and SOD2 protein expression in ischemic retina. Values are mean ± SD (n = 4 retinas/group). *Significant at p<0.05 compared with non-ischemic contralateral control retina or vehicle-treated ischemic retina.

Article Snippet: The membrane was blocked with 5% nonfat dry milk and 0.1% Tween-20 in PBS, incubated with monoclonal mouse anti-GFAP (1∶3000; Sigma), goat polyclonal anti-Tfam antibody (1∶1000; Santa Cruz Biotechnology), mouse monoclonal anti-total OXPHOS complex antibody (containing a mixture of antibodies to COXI-IV and ATP synthase, 1∶3000; Invitrogen), rabbit polyclonal anti-Bax antibody (1∶500; Santa Cruz Biotechnology), rabbit polyclonal anti-Bcl-xL antibody (1∶1000; Cell Signaling, Danvers, MA), mouse monoclonal anti-phosphorylated Bad (pBad, 1∶2000; Cell Signaling), mouse monoclonal anti-NR1 antibody (1∶1000; BD Pharmingen, San Diego, CA), rabbit polyclonal anti-NR2A antibody (1∶500; Millipore), rabbit polyclonal anti-voltage-dependent anion channel (VDAC) antibody (Porin, 1∶1000; Calbiochem, La Jolla, CA) and mouse monoclonal anti-actin antibody (1∶5000, Millipore, Billerics, MA).

Techniques: Injection, Expressing, Labeling, Activation Assay