MA1-2014

antibody from Invitrogen Antibodies

Targeting: GRIN2B GluN2B, NMDAR2B

Provider product page for MA1-2014

Western blot

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [27]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [1]
Other assay [12]

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Product number: MA1-2014 - Provider product page
Provider: Invitrogen Antibodies
Product name: NMDAR2B Monoclonal Antibody (NR2B)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA1-2014 detects the N-methyl-D-aspartate (NMDA) receptor type 2B in human and mouse samples. MA1-2014 has been used successfully in Western blot, IHC (P) and immunocytochemistry procedures. In Western blot analysis of mouse brain tissue this antibody detects a ~166 kDa protein representing NMDA receptor type 2B. The MA1-2014 immunogen is a recombinant protein composed of amino acid residues 934-1457 of the rat NMDA receptor type 2B.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: NR2B
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

GRIN2B protein structure - MA1-2014 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Design of lentiviral vectors for NR2B manipulation in LMAN. ( a ) Diagram of the eGFPhNR2B vector to overexpress human NR2B . ( b ) Diagram of the eGFP vector for control injections. ( c ) Western blots showing no human NR2B protein in HEK293FT cells without transfection (Lane 1), and greater NR2B protein (* ~180 kD band) in cells transfected with the eGFPhNR2B plasmid (possible degraded or unfinished NR2B protein product is detected as an additional lower band) (Lane 2). ( d ) HEK293FT cells transfected with the eGFP plasmid show GFP-expressing cells labeled using a GFP antibody (green). ( e ) Cells transfected with the eGFP plasmid stained with an anti-NR2B antibody show no labeled cells. ( f ) Merged image of ( d ) and ( e ). ( g ) Cells transfected with the eGFPhNR2B plasmid show GFP-expressing cells (localized in cell membranes) labeled using a GFP antibody (green). ( h ) Cells transfected with the eGFPhNR2B plasmid show NR2B-expressing cells (localized in cell membranes) stained with anti-NR2B antibody (red). ( i ) Merged image of ( g ) and ( h ) showing complete overlap of the human NR2B tagged with eGFP (yellow). ( j ) Darkfield view of a brain section showing the injection site in LMAN. ( k ) Cells expressing GFP-tagged (green) human NR2B ~3 weeks after injection of the eGFPhNR2B lentivirus in LMAN. ( l ) Darkfield photograph of coronal sections showing drawings of LMAN and part of nidopallium that was microdissected for protein analysis. ( m ) Western blots

Supportive validation

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Experimental details: Figure 3 Immunostaining of NR2B protein in LMAN cells. ( a ) Quantification showing higher levels of labeled cells in birds injected with the eGFPhNR2B lentivirus in LMAN (blue) compared to control birds with injections of the eGFPhNR2B lentivirus in nidopallium or the eGFP lentivirus in LMAN (red). Control-injected animals did not differ in number of labeled cells (p = 0.73, two sample Wilcoxon test). ( b ) Examples of labeled LMAN cells in eGFP-injected animals (i), and in eGFPhNR2B-injected animals (ii). Scale bar, 200 mum.

Supportive validation

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Experimental details: Figure 2 Expression of NR1, NR2A, and NR2B subunits of glutamate NMDARs in mouse PAG: Co-precipitation with alpha2(delta1), alpha2(delta2), and (alpha2)delta1 proteins. ( A ) PAG P2 fractions enriched in synaptosomes from the CD1 WT, CD1 sigma1R -/- , 129 WT, and 129 HINT1 -/- mice of Figure 1 B,C, were used. About 60 mug protein/lane was resolved by SDS-PAGE and examined in Western blots that were probed with antibodies against NR1, NR1 C1, NR2A, and NR2B NMDAR subunits, as described in Methods. The assay was repeated twice with comparable outcomes. Immunoprecipitation assays on CD1 WT mice ( n = 12): PAG and SC membranes from CD1 WT mice were solubilized with 1% NP-40 and incubated overnight at 4 degC with affinity-purified biotinylated IgGs raised against the ( B ) NR1, ( C ) NR2A, or ( D ) NR2B subunits. Protein complexes were immunoprecipitated (IP) with streptavidin agarose, resolved by SDS-PAGE and visualized in Western blots. The expected size of the alpha2(delta1) and alpha2(delta2) is approximately 100 kDa; however, the bands detected usually appeared as a doublet of 100-140 kDa. ( C , D ), The material associated with the NR2A or NR2B subunits was subjected to deglycosylation with PNGase F, which depleted the alpha2(delta) 140 kDa band in favor of a 100 kDa band. In SC synaptosomes, (alpha2)delta1-2 immunosignals were enriched at NR2A/B subunits (each lane was loaded with solubilized spinal cord tissue from a single CD1 WT mouse, for more details see Section 2 ). I

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Extrasynaptic GluN2B S1303 phosphorylation and interaction with DAPK1 are elevated in the YAC128 brain. Cortical or striatal tissues were subjected to subcellular fractionation to yield (A,C) synaptic (PSD), (B) cortical extrasynaptic (non-PSD), or (D) striatal ""non-synaptic"" membrane fractions, which were run by SDS-PAGE and Western blotting for GluN2B expression and S1303 phosphorylation ( n = 6-14 biological replicates, two technical replicates each; Student's t -test, * p < 0.05, ** p < 0.01). (E) DAPK1 was immunoprecipitated from WT and YAC128 cortical lysates, followed by the detection of co-immunoprecipitated GluN2B by Western blot. All sample blot images in panel (E) are cropped from the same Western blot membrane which contained multiple biological replicates run side-by-side. Data are normalized to WT values ( n = 14 biological replicates, Student's t -test, * p < 0.05, ** p < 0.01).

Supportive validation

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Experimental details: Figure 3 Low-dose memantine normalizes cortical DAPK1 activation and extrasynaptic pS1303 levels in YAC128 mice. Cortical tissues from 1-month-old WT and YAC128 mice treated with low-dose memantine from conception were processed to obtain (A) total or (B) extrasynaptic (non-PSD) membrane protein fractions. Samples were processed for DAPK1 or GluN2B protein expression and phosphorylation levels by Western blot. Data are normalized to WT H 2 O values ( n = 8 biological replicates, two technical replicates each; two-way ANOVA with Bonferroni post hoc analysis, * p < 0.05, ** p < 0.01; Student's t -test, # p < 0.05; two-way ANOVA interaction * p < 0.05 for DAPK1 protein level and pS308, two-way ANOVA interaction ** p < 0.01 for pS1303).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 DAPK1 inhibition normalizes extrasynaptic GluN2B phosphorylation and DAPK1 protein levels in the YAC128 model. (A) Corticostriatal primary cultures from WT and YAC128 embryos were treated at DIV21 with DMSO (0.1%) or a DAPK1 inhibitor (DKI, 10 muM) for 1 h followed by lysis in a gentle buffer to solubilize non-synaptic protein. Lysates were processed by Western blot to detect GluN2B, pS1303, and DAPK1 levels. Data are normalized to WT DMSO values ( n = 8 biological replicates; two-way ANOVA with Bonferroni post hoc analysis, * p < 0.05; Student's t -test, # p < 0.05). (B) Four-week-old YAC128 mice were treated intranasally with 50 nmol of DAPK1 inhibitor (DKI; TC-DAPK6) or vehicle control (VEH). After 6 h, cortical extrasynaptic fractions were isolated to measure GluN2B, pS1303, and DAPK1 levels ( n = 9 biological replicates, two technical replicates each; Student's t -test, * p < 0.05). (C) Quantitative measurement of DAPK1-FLAG protein turnover using bio-orthogonal labeling, FLAG immunoprecipitation, CLICK chemistry, and detection of AHA/Streptavidin (Strep)-labeled DAPK1 in transiently transfected COS-7 cells treated with DMSO or 10 muM DKI (TC-DAPK6) for 24 h ( n = 3 for 8 h and 10 h chase time points, n = 7 for all other chase time points; two-way ANOVA ** p < 0.01 for DKI treatment effect; Sidak's multiple comparisons test, ** p < 0.01).