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: