glun2b n terminus  (Alomone Labs)

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: The GluN2B CaMKII Site Is Dispensable for the Developmental Switch In Vitro (A) Schematic of the GluN2B ΔCaMKII amino acid changes. (B and C) Altered GluN2B phosphorylation in GluN2B ΔCaMKII/ΔCaMKII neurons. Cortical neuronal extracts were prepared and analyzed by western blot with the indicated antibodies, normalized to total GluN2B. (B) shows quantitation (mean ± SEM here and throughout), and (C) shows an example. ∗ p < 0.05 versus GluN2B +/+ (n = 4). (D) NMDAR current density at the indicated ages for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2B WT/WT : n = 14 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2B ΔCaMKII/ΔCaMKII : n = 15 [DIV7 or DIV8], n = 17 [DIV14–18]). (E) Extrasynaptic NMDAR currents were measured in GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons (n = 8 per genotype). (F and G) GluN2A and GluN2B expression analyzed by western blot at DIV8 and DIV16 for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. (F) shows quantitation and (G) shows an example. ∗ p < 0.05 versus DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (H and I) Neocortices of GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII mice were subjected to immunoprecipitation with an antibody to the N terminus of GluN2B, followed by analysis of GluN2B and CaMKIIα content. (H) shows an example and (I) shows quantitation. ∗ p < 0.05; n = 8 per genotype. (J) Percentage blockade of NMDAR currents by ifenprodil (3 μM) at the indicated stages for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2B WT/WT : n = 14 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2B ΔCaMKII/ΔCaMKII : n = 15 [DIV7 or DIV8], n = 17 [DIV14–18]). (K and L) Percentage potentiation of NMDAR currents by 100 μM spermine was measured at the indicated stages for GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2B WT/WT : n = 13 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2B ΔCaMKII/ΔCaMKII : n = 18 [DIV7 or DIV8], n = 14 [DIV14–18]). (K) shows quantitation and (L) shows example traces; scale bar: 200 pA/2 s. (M and N) Neocortical extracts from P14 and P28 mice were analyzed for GluN2A expression, normalized to β-actin. (M) shows quantitation and (N) shows an example. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (O and P) Post-synaptic density (PSD) extracts from P14 and P28 mice of the indicated genotype were analyzed for GluN2A and GluN2B expression, normalized to β-actin, and the ratio calculated. (O) shows quantitation and (P) shows an example. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8).

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: In Vitro, Western Blot, Quantitation Assay, Expressing, Immunoprecipitation

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: The GluN2B CaMKII Binding Site Is Dispensable for Theta Burst LTP (A–C) Activity-dependent signaling to ERK1/2 does not require GluN2B CTD-specific sequences. DIV9 cortical neurons of the indicated genotypes were treated with TTX (500 nM), KN-62 (10 μM), or MK-801 (10 μM) for 1 hr, after which protein extracts were made and subjected to western blot analysis for phospho-ERK1/2 levels, normalized to total ERK1/2. (A) shows quantitation and (B) and (C) show example blots. ∗ p < 0.05 two-way ANOVA plus Dunnett’s post hoc test. No genotype-dependent effects were observed (p = 0.39) or genotype-drug interactions (p = 0.16; n = 8 GluN2B WT/WT ; n = 4 GluN2B ΔCaMKII/ΔCaMKII ; n = 4 GluN2B 2A(CTR)/2A(CTR) ). (D–F) Cumulative probability plots show amplitude (D) and frequency (E) of miniature EPSCs in GluN2B WT/WT and GluN2B ΔCaMKII/ΔCaMKII slices. Results are from 11 cells from 3 GluN2B WT/WT mice and 13 cells from 3 GluN2B ΔCaMKII/ΔCaMKII mice; p = 0.75 (amplitude); p = 0.94 (frequency); unpaired t test. (F) shows example traces. Scale bar: 10 pA/100 ms. (G) NMDAR:AMPAR EPSC ratios at −80 and +40 mV was measured in GluN2B WT/WT (n = 25; 4 mice) and GluN2B ΔCaMKII/ΔCaMKII (n = 30; 4 mice) neurons (unpaired t test; p = 0.95). (H) Weighted time constant (τw) for the decay of EPSCs recorded in (A) at +40 mV was determined using double exponential fits (unpaired t test; p = 0.63). (I) Example traces. Scale bar: 100 pA/20 ms. (J and K) Theta-burst stimulation elicited pathway-specific LTP of synaptic transmission in hippocampal CA1 area. Normalized magnitude of this potentiation 60–65 min after LTP induction did not differ significantly in GluN2B WT/WT mice (29 slices; n = 9) compared to GluN2B ΔCaMKII/ΔCaMKII (35 slices; n = 11); p = 0.162 (two-way nested ANOVA). (J) shows quantitation of data and (K) shows example traces before and after LTP induction. (K) Traces show example fEPSP traces immediately before and 1 hr after theta-burst stimulation. Scale bar: 0.5 mV/2 ms.

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Binding Assay, Activity Assay, Western Blot, Quantitation Assay, Transmission Assay

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: Distinct GluN2 CTDs Are Not Required for the 2B-to-2A Switch (A) Schematic illustrating the C-terminal domain exchange in the GluN2A 2B(CTR)/2B(CTR) mouse. (B) NMDAR current density in GluN2A WT/WT (n = 30) and GluN2A 2B(CTR)/2B(CTR) (n = 24) neurons, recorded DIV14–18. (C and D) Percentage potentiation of NMDAR currents by 100 μM spermine was measured in GluN2A WT/WT and GluN2A 2B(CTR)/2B(CTR) neurons. ∗ p < 0.05 versus DIV7 or DIV8 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (GluN2A WT/WT : n = 19 [DIV7 or DIV8], n = 18 [DIV14–18]; GluN2A 2B(CTR)/2B(CTR) : n = 16 [DIV7 or DIV8], n = 17 [DIV14–18]). (C) shows quantitation of data and (D) shows example traces; scale bar: 200 pA/2 s. (E and F) Neocortical extracts from P14 and P28 mice of the indicated genotype were analyzed for GluN2A expression, normalized to β-actin. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (E) shows quantitation and (F) shows example blots. (G and H) PSD extracts from P14 and P28 mice of the indicated genotype were analyzed for GluN2A and GluN2B expression, normalized to β-actin, and the ratio calculated and scaled such that the ratio at P14 for WT = 1. ∗ p < 0.05 versus P14 of the same genotype; two-way ANOVA plus Sidak’s post hoc test (n = 8). (G) shows quantitation and (H) shows an example blot.

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Quantitation Assay, Expressing

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet: GluN2A Expression Is Sufficient to Displace GluN2B from NMDARs (A–D) Total GluN2B expression was measured in extracts from the neocortex at the indicated stages for the indicated genotypes (n = 8 per genotype). (A) and (B) show quantitation and example blots, respectively, for GluN2A WT/WT versus GluN2A 2B(CTR)/2B(CTR) . (C) and (D) show quantitation and example blots, respectively, for GluN2A WT/WT vs. GluN2B ΔCaMKII/ΔCaMKII . (E–G) Ectopic GluN2A expression is sufficient to displace GluN2B from NMDARs. Young mouse (E and F) and rat (G) neurons at DIV7 were transfected with a control (β-globin) or GluN2A-encoding vector and spermine (100 μM) potentiation of NMDAR currents measured 72 hr later. ∗ p < 0.05 (F: n = 10 of both condition; G: n = 8 control; n = 10 GluN2A). (H) Percentage potentiation of NMDAR currents by spermine (200 μM) in DIV15 or DIV16 rat cortical neurons was measured at the indicated stages for GluN2A +/+ , GluN2A +/− , and GluN2A −/− genotypes. Person r correlation coefficient: −0.9954; p = 0.031 (one-tailed test); n = 12 cells per genotype. (I) Western blot illustrating GluN2A expression in GluN2A +/+ , GluN2A +/– , and GluN2A –/– neurons. (I) A western blot confirming the absence of GluN2A expression in GluN2A −/− and an intermediate expression level in GluN2A +/− neurons.

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Expressing, Quantitation Assay, Transfection, Plasmid Preparation, One-tailed Test, Western Blot

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet:

Article Snippet: anti-GluN2B (N terminus) , Thermo Fisher Scientific , Cat# 71-8600; RRID: AB_2534001.

Techniques: Recombinant