Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a) Schematic diagram showing the locations of stimulating and recording electrodes in the hippocampus and in a CA1 pyramidal neuron for whole cell patch-clamp experiments. (b) Comparison of representative whole-cell patch-clamp recordings of pharmacologically isolated NMDAR EPSCs, normalized to the peak amplitude (in %), from infant, adult and aged mice, illustrating differences in deactivation kinetics. (c) The weighted time constant (τ weighted ) was calculated using the relative contribution of both slow/fast components of NMDAR EPSCs and reflects the overall deactivation kinetics. Results are expressed as the mean ± SEM (Kruskal Wallis followed by an Uncorrected Dunn’s multiple comparisons test using the adult group as reference, **p<0.01, n=28-44). (d) Time course of ifenprodil (5µM) effect on pharmacologically isolated NMDAR EPSC amplitude in CA1 pyramidal neurons, measured by whole-cell patch clamp in infant, adult and aged C57BL/6 wild-type mice. Results are expressed as the mean ± SEM (n=9-12). (e) Traces show NMDAR EPSCs recorded before (CTR) and after 30 min of Ifenprodil 5 µM perfusion (Ifen). (f) GluN2B contribution was calculated as the percentage of change in NMDAR EPSC amplitude after 30 min of ifenprodil perfusion. Results are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, *p<0.05, ****p<0.0001, n=9-12). (g) Schematic representation and immunoblotting analysis of PSD-enriched fractions from the hippocampal tissue of adult C57BL/6 wild-type mice. Membranes were immunoblotted with antibodies for GluN2B, PSD-95 and synaptophysin. PSD-fractions are enriched in PSD-95, whereas non-PSD fractions contain high levels of synaptophysin. (h) Representative western blot of hippocampal lysates subjected to biochemical fractionation to obtain PSD-enriched fractions from infant, adult and aged C57BL/6 mice. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and PSD-95. (i, j) Results from PSD-enriched fractions were normalized with PSD-95 and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, **p<0.01, n=5). (k) Results show the relative GluN2B/GluN2A in PSD-enriched fractions and are expressed as the mean ± SEM (Kruskal Wallis followed by an Uncorrected Dunn’s multiple comparisons test using the adult group as reference, *p<0.05, n=5).

Article Snippet: The following antibodies were used: APP C-terminal Y188 (1:1000, ab32136, Abcam), GluN2A (1:200, sc-136004, Santa Cruz), GluN2B (1:1000, D15B3, Cell Signalling), PSD95 (1:1000, D27E11, Cell signalling), Synaptophysin (1:200, S5768, Merck Millipore), β-actin (1 :1000, sc-47778, Santa Cruz), GAPDH (1:1000, AM4300, Invitrogen).

Techniques: Patch Clamp, Comparison, Isolation, Western Blot, Fractionation

Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a) A slow was calculated as the amplitude of the slow component of NMDAR EPSCs normalized to the total amplitude (%), measured by whole-cell patch-clamp recordings in CA1 hippocampal neurons from infant, adult and aged C57BL/6 mice. Results are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, **p<0.01,****p<0.0001, n=28-44). (b) Representative western of hippocampal lysates from infant, adult and aged C57BL/6 wild-type mice. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and β-actin. (c) Results from blots as shown in (b) from hippocampal lysates were normalized with β-actin and are expressed as the mean ± SEM (Kruskal Wallis test followed by Uncorrected Dunn’s test using the adult group as reference, ****p<0.0001, n=8). (d) Results from blots as shown in (b) from hippocampal lysates were normalized with β-actin and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, **p<0.01, *p<0.05, n=8). (e) Results from blots as shown in (b) from hippocampal lysates show the GluN2B/GluN2A relative ratio and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, ****p<0.0001, n=8). (f) Representative western blot of hippocampal lysates from infant, adult and aged C57BL/6 wild-type mice immunoprecipitated for PSD-95. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and PSD-95. (g, h) Results from blots as shown in (f) from PSD-95 immunoprecipitated samples were normalized with PSD-95 and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, ****p<0.001, n=4). (i) Results from PSD-95 immunoprecipitated samples show the GluN2B/GluN2A relative ratio expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, ****p<0.0001, n=4). (j) Representative western blot of prefrontal cortex human samples (21 to 89 years old). Membranes were immunoblotted with antibodies for GluN2B and GAPDH. (k) Linear regression graph calculated from blots as shown in (j) shows the variation in GluN2B relative levels (normalized with GAPDH) depending on the age of human subjects (n=12). Statistical analysis was performed using Pearson’s correlation (two-tailed p value). Dotted lines represent the 95% confidence intervals. The values obtained for 20–25-year-old subjects were used as reference.

Article Snippet: The following antibodies were used: APP C-terminal Y188 (1:1000, ab32136, Abcam), GluN2A (1:200, sc-136004, Santa Cruz), GluN2B (1:1000, D15B3, Cell Signalling), PSD95 (1:1000, D27E11, Cell signalling), Synaptophysin (1:200, S5768, Merck Millipore), β-actin (1 :1000, sc-47778, Santa Cruz), GAPDH (1:1000, AM4300, Invitrogen).

Techniques: Patch Clamp, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a) Representative western blot of hippocampal PSD-enriched fractions from infant, adult and aged wild-type C57BL/6 mice. Membranes were immunoblotted with antibodies for APP and PSD-95. (b) APP levels were normalized with PSD-95 and are expressed as the mean ± SEM (Kruskal Wallis test followed by Uncorrected Dunn’s test for multiple comparisons using the adult group as reference, ***p<0.001, n=8). (c) Representative western blot of synaptosome fractions from the hippocampi of infant, adult and aged wild-type C57BL/6 mice immunoprecipitated for APP. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and APP. (d) Representative western blot of postmortem brain tissue (prefrontal cortex) from human subjects (adult=22 and aged=89 years old) immunoprecipitated for APP. Membranes were immunoblotted with antibodies for GluN2B and APP. (e) Time course of NMDAR EPSC amplitude measured by whole-cell patch clamp in CA1 pyramidal neurons of infant C57BL/6 wild-type mice during 60 min of incubation with an antibody against the APP C-terminal (Y188). In the control condition, the antibody was heat inactivated (boiled Y188). Results are expressed as the mean ± SEM (n=6-10). The schematic diagram shows the strategy used to mask the APP C-terminal domain - the antibody was added to the intracellular solution in the patch pipette to diffuse into the intracellular space. (f) Traces show NMDAR EPSCs recorded at 20min (baseline) and 60min. The Y188 antibody was inside the patch pipette during the whole course of the experiment (60min). (g) The percentage of Y188-sensitive NMDAR EPSCs was determined for infant, adult and aged C57BL/6 wild-type mice. The effect was calculated comparing the baseline amplitude (15-20 min) with the final amplitude (60 min) and normalized with the control condition (boiled Y188) for each age. Results are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test, ***p<0.001, n=8-14). (h) Time course of NMDAR EPSC amplitude measured by whole-cell patch clamp in CA1 pyramidal neurons of infant C57BL/6 wild-type mice during 90 min of incubation with the Y188 antibody and perfusion with ifenprodil (5µM) at 60-90 min. In the control condition, the antibody was heat inactivated (boiled Y188) (n=1). The schematic diagram shows the strategy used to block APP (Y188 antibody in the patch pipette) and to inhibit GluN2B-NMDAR (ifenprodil perfusion). (i) Traces show NMDAR EPSCs recorded at 20min (baseline), 60min and 90min. The Y188 antibody was inside the patch pipette during the whole course of the experiment (60min), whereas Ifenprodil perfusion occurred from 60 to 90min. (j) The percentage of ifenprodil-sensitive NMDAR EPSCs in infant mice was calculated comparing the amplitude at 60 min with the final amplitude (90 min). The effect of ifenprodil on NMDAR EPSCs was calculated in neurons without antibody incubation (No antibody, used as reference), incubated with the Y188 antibody (Y188) or the heat-inactivated antibody (boiled Y188) for 90 min. Results are expressed as the mean ± SEM (One-way ANOVA followed by Tukey’s multiple comparisons test, **p<0.01, *p<0.05, n= 4-6).

Article Snippet: The following antibodies were used: APP C-terminal Y188 (1:1000, ab32136, Abcam), GluN2A (1:200, sc-136004, Santa Cruz), GluN2B (1:1000, D15B3, Cell Signalling), PSD95 (1:1000, D27E11, Cell signalling), Synaptophysin (1:200, S5768, Merck Millipore), β-actin (1 :1000, sc-47778, Santa Cruz), GAPDH (1:1000, AM4300, Invitrogen).

Techniques: Western Blot, Immunoprecipitation, Patch Clamp, Incubation, Control, Transferring, Blocking Assay

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Ethanol place preferences is associated with the expression of mRNA from GluN2A but not from GluN1 subunit in the NAc. A , Schematic of the experimental design. B , Place preference elicited by the administration of 70 nmol of ethanol in the pVTA. Data are mean ± SEM represented as preference score (test minus pretest time spent in ethanol-paired compartment). * denotes significant differences between means (p<0.01, t-Test). C , Diagram of brain coronal sections indicating in blue the area of all microinjections in pVTA. D , Expression of mRNA from NMDA subunits GluN1 and GluN2A in NAc after ethanol induced CPP. Data are mean ± SEM represented as number of dots (mRNA molecules) per cell. * denotes significant differences between means (p<0.05). E , Expression of mRNA from NMDA subunits GluN1 and GluN2A in hippocampus after ethanol induced CPP. Data are mean ± SEM represented as number of dots (mRNA molecules) per cell.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Expressing

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative pictures of the compilation files obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in NAc (left) and hippocampus (right) after ethanol induced CPP. Squares delimit the area of the amplified pictures shown in for the NAc and in for the hippocampus. Abbreviatures: Aca , anterior commissure; CA3 , field CA3 of hippocampus; DG , dentate gyrus; CA1 , field CA1 of hippocampus.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: In Situ Hybridization, Amplification

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative amplified pictures obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in NAc. Amplified pictures from one animal of each group (aCSF on the left and ethanol 70 nmol on the right) were selected.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Amplification, In Situ Hybridization

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative amplified pictures obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in hippocampus. Amplified pictures from one animal of each group (aCSF on the left and ethanol 70 nmol on the right) were selected.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Amplification, In Situ Hybridization

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Simplified representation of the dose-dependent ethanol effect on place preference or place aversion and its hypothesized mechanism. The administration of low doses of ethanol into the VTA does not induce preference or aversion for the drug paired compartment. Medium doses elicit CPP, presumably through an increase of the ethanol metabolized fraction. In our hypothesis and based in previous literature, the ethanol metabolite, salsolinol, would activate MORs, decreasing GABA release and finally increasing DA release in the NAc by a disinhibition of VTA DA neurons. The expression of ethanol induced CPP also results in an increase of mRNA form GluN2A in the NAc. Moreover, the blockade of the MORs in the VTA inhibits the acquisition of this ethanol induced CPP. However, the administration of high doses results in the development of CPA. In this case, the effect of the non-metabolized fraction would predominate and the inhibition of the VTA DA neurons would result in a decrease of DA release in the NAc. In orange GABA terminals and in purple VTA DA neurons. MOR: mu opioid receptor; GABAR: gamma-amino butyric acid (GABA) receptor; NAc: nucleus accumbens; VTA: ventral tegmental area; DA: dopamine; GLUN2A: NMDA receptor subunit 2A.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Expressing, Inhibition

Journal: Nature neuroscience

Article Title: A neuronal role for SNAP-23 in postsynaptic glutamate receptor trafficking

doi: 10.1038/nn.2488

Figure Lengend Snippet: ( a ) Targeting strategy to remove Snap-23 exon 2 (E2) containing the initiator ATG. The structures of the wild-type Snap-23 gene and the targeting construct are shown. Exons E1 to E5 are represented as black boxes. Using a genomic clone harboring the Snap-23 gene, a targeting vector containing the neomycin resistance gene flanked by loxP sites and the TK gene was generated. Following homologous recombination in ES cells, targeted heterozygous mice containing targeted allele were obtained. Mice harboring the Snap-23 E2-deleted allele were generated by breeding with EIIa-Cre transgenic mice. ( b ) Southern blot analysis of genomic DNA isolated from tails of Snap-23 E2-targeted mice. EcoRI-digested genomic DNA was hybridized with the 5′ probe. ( c ) Genomic PCR to detect the targeted and wild-type alleles from tail DNA of E2-targeted mice. ( d ) Expression of SNAP-23 was analyzed by immunoblotting whole brain lysates of SNAP-23 heterozygous (Het) mice and wild-type (WT) littermates. ( e ) Expression of glutamate receptors in the P2 crude synaptosome fraction from hippocampus of SNAP-23 Het and WT littermates. ( f ) Surface expression of glutamate receptors was analyzed using a surface biotinylation assay in primary cortical neurons from SNAP-23 Het and WT littermates. Surface receptors were isolated by precipitation using Streptavidin-agarose beads and immunoblotted with the indicated antibodies. ( g ) Quantitation of the immunoblots was performed by measuring the band intensity of the biotinylated fraction compared with the intensity of total input using ImageJ software. Graphs represent means ± s.e.m. *p<0.01 (n = 3–5) (p=0.0077 for NR2A, 0.0097 for NR2B, 0.0095 for NR1, 0.4130 for GluR1, 0.7772 for GluR2, 0.1706 for GABA(A) α1, and 0.4740 for mGluR7).

Article Snippet: The following antibodies were purchased from commercial sources: PSD-95 6G6-1C9, GluR2 6C4 and polyclonal, GluR1 polyclonal, NR1 54.1, NR2A polyclonal, mGluR7a polyclonal (Millipore Corporation, Billerica, MA); NR1 R1JHL (Affinity BioReagents, Golden, CO); Synaptophysin SVP-38, microtubule-associated protein 2 (MAP2) HM-2, alpha-tubulin (Sigma, St. Louis, MO); pan-Shank N23B/49 (NeuroMab, Davis, CA); SNAP-25 SMI 81 (Covance, Berkeley, CA); Synaptobrevin2/VAMP-2 69.1, Gephyrin mAb7a (Synaptic Systems, Göttingen, Germany), GABA(A) α1 (Alomone Labs Ltd, Jerusalem, Israel).

Techniques: Construct, Plasmid Preparation, Generated, Homologous Recombination, Transgenic Assay, Southern Blot, Isolation, Expressing, Western Blot, Surface Biotinylation Assay, Quantitation Assay, Software

Journal: Nature neuroscience

Article Title: A neuronal role for SNAP-23 in postsynaptic glutamate receptor trafficking

doi: 10.1038/nn.2488

Figure Lengend Snippet: ( a ) Primary hippocampal neurons (DIV 5–7) were transduced with scrambled, SNAP-23, or SNAP-25 shRNA lentivirus for 7 days. Surface expression of glutamate receptors was evaluated using a surface biotinylation assay. ( b ) Quantitation of the immunoblots was performed by measuring the band intensity of the biotinylated fraction compared with the band intensity of total input using ImageJ software. Graphs represent means ± s.e.m. **p<0.01, *p<0.05 (n = 5) [p=0.0004 (NR2A), 0.0078 (NR2B), 0.0004 (NR1), 0.0103 (GluR1), 0.0176 (GluR2) for SNAP-23 shRNA; p=0.4526 (NR2A), 0.4727 (NR2B), 0.9965 (NR1), 0.7723 (GluR1), 0.9977 (GluR2) for SNAP-25 shRNA compared with scrambled shRNA].

Article Snippet: The following antibodies were purchased from commercial sources: PSD-95 6G6-1C9, GluR2 6C4 and polyclonal, GluR1 polyclonal, NR1 54.1, NR2A polyclonal, mGluR7a polyclonal (Millipore Corporation, Billerica, MA); NR1 R1JHL (Affinity BioReagents, Golden, CO); Synaptophysin SVP-38, microtubule-associated protein 2 (MAP2) HM-2, alpha-tubulin (Sigma, St. Louis, MO); pan-Shank N23B/49 (NeuroMab, Davis, CA); SNAP-25 SMI 81 (Covance, Berkeley, CA); Synaptobrevin2/VAMP-2 69.1, Gephyrin mAb7a (Synaptic Systems, Göttingen, Germany), GABA(A) α1 (Alomone Labs Ltd, Jerusalem, Israel).

Techniques: Transduction, shRNA, Expressing, Surface Biotinylation Assay, Quantitation Assay, Western Blot, Software

Journal: Biochimica et biophysica acta

Article Title: Specificity Protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B

doi: 10.1016/j.bbamcr.2013.07.002

Figure Lengend Snippet: In vivo ChIP assays for Sp4 interaction with NMDA receptor subunits in mouse visual cortical tissue. Chromatin was precipitated with anti Sp4 antibodies (Sp4 IP lane), anti-nerve growth factor receptor p75 antibody (negative control, NGFR IP lane) or no antibody (negative control, no Ab lane). Control reactions for PCR were performed with 0.5% (input 0.5% IP lane) and 0.1% (input 0.1% IP lane) of input chromatin. GM3 synthase and Neurotrophin 3 were used as positive controls, and β-actin was used as a negative control. Results indicate interactions of Sp4 with Grin1, Grin2a, and Grin2b but not with Grin2c.

Article Snippet: Subsequent to blocking, blots were incubated in primary antibodies against Sp1 (1:1000; Santa Cruz), Sp3 (1:1000; Santa Cruz), Sp4 (1:1000; Santa Cruz), GluN1 (1:1000; Millipore Chemicon, Billerica, MA, USA), GluN2A (1:600; 19953-1-AP, ProteinTech, Chicago, IL, USA), and GluN2B (1:1000; 1498-NR2B, PhosphoSolutions, Aurora, CO, USA). β-actin (1:5000; Sigma) served as a loading control for cytoplasmic extracts and for Sp1, Sp3, and Sp4 shRNA and over-expression samples.

Techniques: In Vivo, Negative Control, Control

Journal: Biochimica et biophysica acta

Article Title: Specificity Protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B

doi: 10.1016/j.bbamcr.2013.07.002

Figure Lengend Snippet: Site-directed mutational analysis of promoters of wild type (wt) and those with mutated Sp4 binding site (mut) for Grin1, Grin2a, and Grin2b genes in N2a cells. Mutating the Sp4 binding sites on Grin1, Grin2a, and Grin2b genes resulted in significant decreases in luciferase activity. KCl depolarization significantly increased promoter activity in all wild types, but not in the Grin1, Grin2a, and Grin2b promoters with mutated Sp sites. N = 6 for each construct. ***= P < 0.001; X = NS. All mutants and wild type + KCl are compared to the wild type. All mutant + KCl are compared to mutants.

Article Snippet: Subsequent to blocking, blots were incubated in primary antibodies against Sp1 (1:1000; Santa Cruz), Sp3 (1:1000; Santa Cruz), Sp4 (1:1000; Santa Cruz), GluN1 (1:1000; Millipore Chemicon, Billerica, MA, USA), GluN2A (1:600; 19953-1-AP, ProteinTech, Chicago, IL, USA), and GluN2B (1:1000; 1498-NR2B, PhosphoSolutions, Aurora, CO, USA). β-actin (1:5000; Sigma) served as a loading control for cytoplasmic extracts and for Sp1, Sp3, and Sp4 shRNA and over-expression samples.

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Mutagenesis

Journal: Biochimica et biophysica acta

Article Title: Specificity Protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B

doi: 10.1016/j.bbamcr.2013.07.002

Figure Lengend Snippet: Effect of RNA interference-mediated silencing of Sp1, Sp3, or Sp4 on the expression of the NMDA receptor subunit genes. (A) Real-time PCR revealed a down-regulation of Sp1, Sp3, and Sp4 transcripts in N2a cells transfected with Sp1, Sp3, and Sp4 shRNA, respectively. N = 6. (B) mRNA levels of Grin1, Grin2a, and Grin2b were decreased with Sp4 shRNA but not with Sp3 or Sp1 shRNA. N = 6. (C–D) Western blots revealed a down-regulation of Sp4, Sp3, and Sp1 proteins in Sp4, Sp3, and Sp1 shRNA-transfected N2a cells, respectively. β-actin served as loading control and a representative blot is shown. N = 3. (E–F) Silencing of Sp4 reduced the protein levels of GluN1, GluN2A, and GluN2B, whereas silencing of Sp1 and Sp3 did not significantly change these subunit levels. β-actin served as a loading control. N = 3. (G) Primary neurons transfected with Sp1, Sp3, or Sp4 shRNA showed decreases in Sp1, Sp3, and Sp4 transcript, respectively. N = 3. (H) mRNA levels of Grin1, Grin2a, and Grin2b were decreased with Sp4 shRNA but not with Sp3 or Sp1 shRNA in primary neurons. Grin2c levels did not decrease with Sp1, Sp3, or Sp4 shRNA in primary neurons. N = 3. *= P < 0.01, **= P < 0.01, and ***= P < 0.001.

Article Snippet: Subsequent to blocking, blots were incubated in primary antibodies against Sp1 (1:1000; Santa Cruz), Sp3 (1:1000; Santa Cruz), Sp4 (1:1000; Santa Cruz), GluN1 (1:1000; Millipore Chemicon, Billerica, MA, USA), GluN2A (1:600; 19953-1-AP, ProteinTech, Chicago, IL, USA), and GluN2B (1:1000; 1498-NR2B, PhosphoSolutions, Aurora, CO, USA). β-actin (1:5000; Sigma) served as a loading control for cytoplasmic extracts and for Sp1, Sp3, and Sp4 shRNA and over-expression samples.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, shRNA, Western Blot, Control

Journal: Biochimica et biophysica acta

Article Title: Specificity Protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B

doi: 10.1016/j.bbamcr.2013.07.002

Figure Lengend Snippet: Effect of Sp1, Sp3, and Sp4 over-expression on the transcript and protein levels of NMDA receptor subunit genes. (A) Real-time PCR revealed an up-regulation of Sp1, Sp3, and Sp4 transcripts in N2a cells transfected with Sp1, Sp3, and Sp4 over-expression vectors, respectively. N = 6. (B) mRNA levels of Grin1, Grin2a, and Grin2b were increased with Sp4 over-expression but not with Sp3 or Sp1 over-expression. N = 6. (C-D) Western blots revealed an up-regulation of Sp4, Sp3, and Sp1 protein with Sp4, Sp3, and Sp1 over-expression, respectively, in N2a cells. β-actin served as a loading control and a representative blot is shown. N = 3. (E–F) Over-expression of Sp4 increased protein levels of GluN1, GluN2A, and GluN2B, whereas over-expression of Sp1 and Sp3 did not significantly change these subunit levels. β-actin served as a loading control. N = 3. (G) Primary neurons transfected with Sp1, Sp3, or Sp4 over-expression showed increases in Sp1, Sp3, and Sp4 transcripts, respectively. N = 3. (H) mRNA levels of Grin1, Grin2a, and Grin2b were increased with Sp4 over-expression but not with Sp3 or Sp1 over-expression in primary neurons. Grin2c levels did not increase with Sp1, Sp3, or Sp4 overexpression in primary neurons. N = 3. *= P < 0.01, **= P < 0.01, and ***= P < 0.001.

Article Snippet: Subsequent to blocking, blots were incubated in primary antibodies against Sp1 (1:1000; Santa Cruz), Sp3 (1:1000; Santa Cruz), Sp4 (1:1000; Santa Cruz), GluN1 (1:1000; Millipore Chemicon, Billerica, MA, USA), GluN2A (1:600; 19953-1-AP, ProteinTech, Chicago, IL, USA), and GluN2B (1:1000; 1498-NR2B, PhosphoSolutions, Aurora, CO, USA). β-actin (1:5000; Sigma) served as a loading control for cytoplasmic extracts and for Sp1, Sp3, and Sp4 shRNA and over-expression samples.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Control

Journal: Biochimica et biophysica acta

Article Title: Specificity Protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B

doi: 10.1016/j.bbamcr.2013.07.002

Figure Lengend Snippet: Effect of increased or decreased neuronal activity on Sp factors and on target genes in the presence of Sp1, Sp3, or Sp4 silencing or over-expression. (A) N2a cells treated for 5 h with 20 mM KCl revealed an up-regulation of all transcripts as compared to controls. In the presence of Sp4 silencing, 5 h treatment with 20 mM KCl failed to up-regulate the transcripts of Grin1, Grin2a, and Grin2b, but had no effect on Grin2c. Sp3 and Sp1 silencing did not prevent KCl-induced up-regulation of Grin1 and Grin2a-c subunits. N = 6. (B) N2a cells treated for 3 days with 0.4 μM TTX revealed a down-regulation of Grin1 and Grin2a-c subunits as compared to controls. Over-expression of Sp4 rescued the down-regulation of the Grin1, Grin2a, and Grin2b transcripts, but not that of Grin2c. Over-expression of Sp1 or Sp3 did not rescue the down-regulation of Grin1 and Grin2a-c transcripts seen with KCl treatment. N = 6. (C–D) KCl-induced activity increased protein levels of Sp4, whereas TTXinduced impulse blockade decreased Sp4 protein levels in primary neurons. N = 3. (E–F) Increased neuronal activity led to an increase in the nuclear Sp4 but not cytoplasmic Sp4. Nuclear and cytoplasmic levels of Sp1 and Sp3 did not change significantly. β-actin served as a loading control and indicated no cytoplasmic contamination of the nuclear fraction. NeuN was present only in the nucleus and indicated no nuclear contamination of the cytoplasmic fraction. N = 3. *= P < 0.01, **= P < 0.01, and ***= P < 0.001; (A–B) ### = P < 0.001 and X = non-significant when compared to KCl- or TTX-treated samples

Article Snippet: Subsequent to blocking, blots were incubated in primary antibodies against Sp1 (1:1000; Santa Cruz), Sp3 (1:1000; Santa Cruz), Sp4 (1:1000; Santa Cruz), GluN1 (1:1000; Millipore Chemicon, Billerica, MA, USA), GluN2A (1:600; 19953-1-AP, ProteinTech, Chicago, IL, USA), and GluN2B (1:1000; 1498-NR2B, PhosphoSolutions, Aurora, CO, USA). β-actin (1:5000; Sigma) served as a loading control for cytoplasmic extracts and for Sp1, Sp3, and Sp4 shRNA and over-expression samples.

Techniques: Activity Assay, Over Expression, Control

Journal: Biochimica et biophysica acta

Article Title: Specificity Protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B

doi: 10.1016/j.bbamcr.2013.07.002

Figure Lengend Snippet: Aligned partial sequences of Grin1, Grin2a, and Grin2b promoters from mouse, rat, and human showed conserved Sp binding sites.

Article Snippet: Subsequent to blocking, blots were incubated in primary antibodies against Sp1 (1:1000; Santa Cruz), Sp3 (1:1000; Santa Cruz), Sp4 (1:1000; Santa Cruz), GluN1 (1:1000; Millipore Chemicon, Billerica, MA, USA), GluN2A (1:600; 19953-1-AP, ProteinTech, Chicago, IL, USA), and GluN2B (1:1000; 1498-NR2B, PhosphoSolutions, Aurora, CO, USA). β-actin (1:5000; Sigma) served as a loading control for cytoplasmic extracts and for Sp1, Sp3, and Sp4 shRNA and over-expression samples.

Techniques: Binding Assay

Journal: Biochimica et biophysica acta

Article Title: Specificity Protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B

doi: 10.1016/j.bbamcr.2013.07.002

Figure Lengend Snippet: A mechanistic scheme of transcriptional co-regulation by Sp4, NRF-1, and NRF-2. The three factors co-regulate Grin1 and Grin2b in a concurrent and parallel (same direction) manner, but only Sp4 regulates Grin2a, hence via a complementary mechanism.

Article Snippet: Subsequent to blocking, blots were incubated in primary antibodies against Sp1 (1:1000; Santa Cruz), Sp3 (1:1000; Santa Cruz), Sp4 (1:1000; Santa Cruz), GluN1 (1:1000; Millipore Chemicon, Billerica, MA, USA), GluN2A (1:600; 19953-1-AP, ProteinTech, Chicago, IL, USA), and GluN2B (1:1000; 1498-NR2B, PhosphoSolutions, Aurora, CO, USA). β-actin (1:5000; Sigma) served as a loading control for cytoplasmic extracts and for Sp1, Sp3, and Sp4 shRNA and over-expression samples.

Techniques: