Journal: PLoS Biology

Article Title: Neuregulin and BDNF Induce a Switch to NMDA Receptor-Dependent Myelination by Oligodendrocytes

doi: 10.1371/journal.pbio.1001743

Figure Lengend Snippet: (A) Western blots of control and NRG-treated cocultures for NR1, NR2B and its phosphorylated form (pNR2B), NR2C and its phosphorylated form (pNR2C), NR3A and NR3B, as well as for NR2A and NR2D compared to their respective positive controls of rat cortex (Ctx) and thalamus (Th); β-actin acts as a loading control throughout. (B) Densitometric quantification of subunit protein levels in cocultures (normalized to β-actin) in NRG normalized to the levels in control (NR2A and NR2D levels were undetectable). (C) Western blot of control (Con) and NRG-treated pure DRG cultures for NR3A and (below) densitometric quantification of subunit protein levels (normalized to β-actin and then to control). (D) Western blot for NR1 and NR3A of control (Con) and NRG-treated (for 6 d) pure OPC cultures, treated (+) or not treated (−) with 20 min glutamate (Glu, 100 µM) stimulation every day, with densitometric quantification of subunit protein levels (normalized to β-actin and then to control). The p values over the bars, in (B) from Holm–Bonferroni corrected t tests and in (C and D) from one-sample Student t tests, compare with control; numbers of experiments shown on bars.

Article Snippet: Immunoblots were then incubated overnight at 4°C with goat anti-Akt (Santa Cruz, 1∶1,000), rabbit anti-phosphorylated-Akt Ser473 (Cell Signalling, 1∶1,000), rabbit anti-phosphorylated-ERK1/2 Thr202/Tyr214 (Cell Signalling, 1∶1,000), mouse anti-ERK1/2 (Cell Signalling, 1∶2,000), rabbit anti-MBP (Sigma, 1∶3,000), mouse anti-NR1 (Millipore, 1∶1,000), rabbit anti-NR2A (Millipore, 1∶1,000), rabbit anti-NR2B (Abcam; 1∶1,000), rabbit anti-phosphorylated NR2B Tyr1472 (Millipore, 1∶1,000), rabbit anti-NR2C (Millipore, 1∶1,000), rabbit anti-phosphorylated NR2C S1096 (a kind gift from Katherine W. Roche, NINDS), rabbit anti-NR3A (Millipore, 1∶500), rabbit anti-NR3B (Millipore, 1∶300), rabbit anti-NR2D (Abcam, 1∶300), or mouse anti-β-actin (Sigma, 1∶100,000) in 3% BSA in PBS-T.

Techniques: Western Blot

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:

Journal: Journal of neurochemistry

Article Title: Repeated cocaine enhances ventral hippocampal-stimulated dopamine efflux in the nucleus accumbens and alters ventral hippocampal NMDA receptor subunit expression

doi: 10.1111/jnc.12764

Figure Lengend Snippet: (A) Representative protein bands on immunoblots for NR1 (120 kD), NR2A (180 kD), and NR2B (190 kD) in the ventral hippocampus (examples are from single animals in each treatment group). (B) NMDA subunit protein levels in ventral hippocampal tissue of saline- and cocaine-administered rats. (C) Calculated NR2A:NR2B ratios of rats injected with saline or cocaine for seven days. Repeated cocaine resulted in a reduced NR2A:NR2B ratio in the ventral hippocampus compared with repeated saline (*significant difference between saline and cocaine groups, P < 0.05). Means + SEM are shown. (N=9–10/group)

Article Snippet: After blocking for 60 minutes at room temperature, the membranes were incubated overnight at 4 °C with rabbit anti-NR1, NR2B or NR2A primary antibodies (1:1000; mAb #5704; #4205; #4212; Cell Signaling, Danvers, MA).

Techniques: Western Blot, Saline, Injection