Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A, B ) Hippocampal neurons were subjected to ( A ) chemical LTP (cLTP) or ( B ) chemical LTD (cLTD). DKK3 levels were analyzed in the supernatant (extracell) and cell lysates by western blot. Phospho-GluA1 Ser845 was used as a readout of LTP and LTD induction, and tubulin as a loading control for the cell lysate. Graphs show the levels of DKK3 relative to control and the ratio of extracellular/lysate DKK3 levels (Mann-Whitney Test, n=3 independent cultures for cLTP and Student’s T-test, n=6 independent cultures for cLTD). ( C ) Representative immunoblots show DKK3 levels in the cell lysate and extracellular (extracell) fraction of acute WT hippocampal slices treated with vehicle (Ctrl) or NMDA (cLTD) and/or Brefeldin A (BFA) for 60 min. Actin was used as a loading control in homogenates. Graphs show densiometric quantifications of DKK3 relative to the control condition and the ratio of extracellular/lysate DKK3 levels (Kruskal-Wallis followed by Dunn’s multiple comparisons; n=5 animals). ( D ) Immunoblot showing DKK3 is less abundant in the extracellular fraction when compared to the cell lysate fraction. Representatives immunoblot of J20 brain slices treated with control or APV for 3 hr. Time exposure for obtaining DKK3 chemiluminescent images is indicated. Figure 2—figure supplement 3—source data 1. Uncropped western blot gels. Raw and annotated WB images. The representative western blot images for are indicated within a blue square.

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of hippocampal slices following electrophysiology stained for NeuN (grey, AF-647), GluA1 (blue, AF-488), GluA2 (red, AF-594), and DAPI (green) and corresponding merged images for naïve, cLTP, sLTP, cDEP, and sDEP slices (maximum intensity projection). The dotted white line shown in the merged image represents the region of interest (stratum radiatum, SR) for each slice. Scalebar = 200 µm. ( B ) After normalization to naïve slices, the GluA1/GluA2 ratio in the SR was higher following cLTP compared to sLTP (unpaired Student’s t test, mean difference = 0.277 ± 0.084 times the naïve GluA1/GluA2 ratio, p = 0.022). Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. ( C ) No differences in the GluA1/GluA2 ratio were observed following cDEP compared to sDEP (unpaired Student’s t test, mean difference = −0.002 ± 0.186 times the naïve GluA1/GluA2 ratio, p = 0.993). Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. ( D ) Fluorescence intensity profiles normalized to the maximum fluorescence intensity as a function of distance along the SR (normalized to 1) for GluA1 (left) and GluA2 (right) in naïve slices. Data are means ± SEM from 10 biological replicates. ( E ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cLTP or sLTP. Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. Differences in GluA1 fluorescence intensity along the SR relative to the maxima were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.11, p = 0.239; main effect of LTP type F(1,5) = 9.43, p = 0.028). No statistically significant differences in GluA2 distribution were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.23, p = 0.081, LTP type F(1, 5) = 3.11, p = 0.138). ( F ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cDEP or sDEP. Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. No statistically significant differences were observed between cDEP and sDEP for GluA1 (distance x DEP F(99, 591) = 0.459, p > 0.999, DEP type F(1, 6) = 2.07, p = 0.200) or GluA2 (distance x DEP F(99, 591) = 0.680, p = 0.991, DEP type F(1, 6) = 1.69, p = 0.242) using mixed effects analysis. Data from male (closed circles) and female (open circles) mice combined, 1-2 technical replicates per biological replicate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Confocal Microscopy, Staining, Fluorescence

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) GluA1 normalized to naïve hippocampal slices following cDEP and sDEP in control conditions (black), in the presence of 100 µM 7-CK (purple) and in the presence of 50 µM APV (blue). DEP x treatment F(2, 15) = 0.240, p = 0.790. ( B ) GluA2 normalized to naïve slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.220, p = 0.805. ( C ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.583, p = 0.571, main effect of DEP type F(1, 15) = 12.27, p = 0.003. Post-hoc cDEP+7-CK vs. sDEP+7-CK least squares (LS) mean difference = 1.3 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.034; cDEP vs. sDEP LS mean difference = 1.4 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.021. ( D ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 1.62, p = 0.231, main effect of DEP type (F(1, 15) = 5.88, p = 0.028) and drug treatment (F(2, 15) = 6.57, p = 0.0089). Post-hoc cDEP+7CK vs. cDEP+APV LS mean difference = 3.7 ± 1.4 times naïve pGluA1 S845/GluA1 expression, p = 0.054; sDEP vs. sDEP+7-CK LS mean difference = −4.4 ± 1.6 times naïve pGluA1 S845/GluA1 expression, p = 0.043; cDEP vs. sDEP LS mean difference = 4.3 ± 1.5 times naïve pGluA1 S845/GluA1 expression, p = 0.012. Data in (A-D) are means ± SEM from 4 (cDEP) and 3 (sDEP) biological replicates. ( E-H ) AMPAR expression and phosphorylation states are unaltered following cDEP in the presence of C1.1 compared to C1.1Scr. ( E ) GluA1 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr (black) versus C1.1 (grey) (p = 0.555). ( F ) GluA2 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr versus C1.1 (p = 0.876). ( G ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.238) ( H ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.067). Data in (E-G) are means ± SEM from 4 biological replicates per group. 1-2 technical replicates per biological replicate. Blots were cut to probe for each protein (see Fig. S5A-C). Each drug treatment condition was normalized to a naïve slice run in the same blot. Statistical comparisons were made using ordinary two-way ANOVA followed by Holm-Šídák post-hoc comparisons within and across drug treatment conditions (A-D) or unpaired Student’s t test (E-H) as appropriate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Full blots stained for GluA1 (top left-hand side of blots), pGluA1 S831 (top right-hand side of blots) and GAPDH (bottom section of blots) after membranes were cut as indicated along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown. Samples were treated during electrophysiology experiments with 7-CK or APV as indicated. ( B ) Full blots stained for GluA2 (top left-hand side of blots), pGluA1 S845 (top right-hand side of blots) and GAPDH (bottom of blots) after blots were cut along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown with corresponding treatments indicated. ( C ) Full blots stained for GluA1 (top left-hand side of left blot), pGluA1 S831 (top right-hand side of left blot), GluA2 (top left-hand side of right blot) and pGluA1 S845 (top right-hand side of right blot). Cropped blots from main are indicated with the corresponding colour-coded boxes. Images of blots stained for pGluA1 S831 and S845 were mirrored in for clarity.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Western blotting of GluA1, GluA2, NR1, and GABA A R ɑ1 in the brain lysates of WT and Ube3a 2xTg mice. GluA1 expression was reduced in Ube3a 2xTg mice. ( B ) Quantitative analysis of GluA1, GluA2, NR1, and GABA A R ɑ1 in WT and Ube3a 2xTg mice. n = 6 animals/group. ( C ) Synaptic expression of GluA1 was identified by colocalized VGluT1 and GluA1 puncta fluorescent signals. Scale bars, 5 μm. ( D ) Density of GluA1 puncta in DIV14 primary neurons prepared from P0 WT or Tg mice. Quantification showed a decrease in density of GluA1 and GluA1/VGluT colocalized puncta in Tg neurons compared to WT neurons. n = 9 WT, n = 8 Ube3a 2xTg. ( E ) c-Fos expression in rat hippocampal neurons infected with either GFP or UBE3A virus. Neurons were treated with either DMSO or 10 µM Glu in the presence of APV + TTX or APV + TTX + CNQX. The nuclei were stained by Hoechst. Scale bars,100 μm. ( F ) Quantification of the percentage of c-Fos positive cells. Glutamate (Glu) treatment induced a significant increase of c-Fos positive neurons in GFP control but not UBE3A-overexpressing condition, which was blocked by AMPAR antagonist CNQX, indicating that the increase was mediated by AMPARs. GFP-TTX + APV: n = 27, GFP-Glu+TTX + APV: n = 24, GFP-Glu+TTX + APV + CNQX: n = 25, GFP + UBE3A-TTX + APV: n = 26, GFP + UBE3A-Glu+TTX + APV: n = 23, GFP + UBE3A-Glu+TTX + APV + CNQX: n = 23 images of 2.25 mm 2 area. Data information: in ( B , D ), unpaired two-tailed t test. In ( F ), two-way ANOVA with Bonferroni’s multiple comparisons test. Mean ± SEM. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns = not significant.

Article Snippet: Protein A-Agarose beads (Santa Cruz Biotechnology; cat. #sc-2001) were added to the lysates along with antibodies against GluA1 N-terminal (Alomone labs, cat. #AGC-004).

Techniques: Western Blot, Expressing, Infection, Virus, Staining, Control, Two Tailed Test

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) GluA1 ubiquitination assay. HEK293 cells were transfected with GluA1 vector and HA-ubiquitin (ubi), together with either a control vector, UBE3A, or the E3 ligase-dead mutant UBE3A C820A for 2 days. GluA1 was immunoprecipitated and probed for ubi. Cell lysates (input) were also probed to detect total protein levels. (B ) GluA1 RNA FISH in DIV10 primary neurons prepared from P0 WT and Tg mice. Scale bars, 15 μm. The dotted circle highlights the nucleus location, which was stained using Hoechst. ( C ) Total intensity of GluA1 mRNA fluorescence was not changed in Tg neurons. n = 10 neurons/group. ( D ) Subcellular distribution of GluA1 mRNA. Cytosolic GluA1 mRNA was reduced, whereas the nuclear GluA1 mRNA was increased in Tg neurons. n = 10 neurons/group. ( E ) qPCR of total GluA1 mRNA in whole brain lysates showed no difference between WT and Tg mice. n = 4 animals/group ( F ) GluA1 mRNA expression was decreased in the cytosolic fraction and increased in the nuclear fraction of brain lysates from Tg mice. n = 3 WT; n = 4 Ube3a 2xTg. Data information: Mean ± SEM. * p < 0.05; ** p < 0.01; ns = not significant. In ( D , F ), two-way ANOVA with Bonferroni’s multiple comparisons test. In ( C , E ), unpaired two-tailed t test.

Article Snippet: Protein A-Agarose beads (Santa Cruz Biotechnology; cat. #sc-2001) were added to the lysates along with antibodies against GluA1 N-terminal (Alomone labs, cat. #AGC-004).

Techniques: Ubiquitin Assay, Transfection, Plasmid Preparation, Control, Mutagenesis, Immunoprecipitation, Staining, Fluorescence, Expressing, Two Tailed Test

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) GluA1 RNA FISH (red) in DIV14 primary rat neurons transfected with either SCR-GFP, shGluA1-GFP to knock down GluA1, or GluA1-GFP to overexpress GluA1. Hoechst stained nucleus. Arrows point to the transfected cells. Scale bars, 25 μm. ( B ) Quantification showed a reduction in the FISH fluorescent intensity of GluA1 mRNA following GluA1 knock down by shRNA; and increased FISH fluorescent intensity of GluA1 mRNA following GluA1 overexpressing. ( C ) Knocking down or overexpressing GluA1 mainly affected GluA1 mRNA level in the cytosol rather than that in the nucleus. Data information: SCR-GFP: n = 35, shGluA1-GFP: n = 36, GluA1-GFP: n = 33. Mean ± SEM. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant. In ( B ), one-way ANOVA with Bonferroni’s multiple comparisons test. In ( C ), two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Protein A-Agarose beads (Santa Cruz Biotechnology; cat. #sc-2001) were added to the lysates along with antibodies against GluA1 N-terminal (Alomone labs, cat. #AGC-004).

Techniques: Transfection, Knockdown, Staining, shRNA

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Top dysregulated candidates mediating mRNA nuclear transport in Ube3a 2xTg brains identified by RNA-seq. ( B , C ) Immunostaining showed that SARNP expression was reduced in Ube3a 2xTg primary mouse neurons. n = 9 neurons/group. Scale bars, 10 μm. ( D ) Protein expression of SARNP in the brain lysates of WT and Ube3a 2xTg mice was analyzed by western blot. ( E ) Quantitative analysis showed a significant reduction of SARNP in Ube3a 2xTg brains. n = 3 animals/group. ( F ) Protein expression of ENY2 in the brain lysates of WT and Ube3a 2xTg mice was analyzed by western blot. ( G ) Quantitative analysis of ENY2 in Ube3a 2xTg brains. n = 3 animals/group. ( H ) WT and Ube3a 2xTg mouse cultures were transfected with GFP, GFP + SARNP, or GFP + ENY2 at DIV10. GluA1 mRNA (red) was probed by RNA FISH at DIV14. The dotted circle indicates the location of nucleus. Hoechst was used to stain the nucleus. Scale bars, 15 μm. ( I ) Quantification of GluA1 mRNA distribution showed that SARNP restoration was able to rescue GluA1 mRNA nuclear retention. n = 7 WT, n = 9 Ube3a 2xTg, n = 11 Ube3a 2xTg+SARNP, n = 11 Ube3a 2xTg + ENY2. ( J ) Primary rat neurons were transfected with either a control vector, or shSARNP-GFP to knock down SARNP at DIV7. Neurons were fixed at DIV10 and processed for GluA1 RNA FISH. The dotted circle indicates the location of nucleus. Hoechst was used to stain the nucleus. Scale bars, 10 μm. ( K ) Quantification of total GluA1 mRNA intensity in the soma. SCR vector: n = 25, shSARNP-GFP: n = 21. ( L ) Quantification of GluA1 mRNA intensity in the nucleus vs. in the soma. SCR vector: n = 25, shSARNP-GFP: n = 21. Data information: Mean ± SEM. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns = not significant. In ( C , E , G , K , L ), unpaired two-tailed t test. In ( I ), two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Protein A-Agarose beads (Santa Cruz Biotechnology; cat. #sc-2001) were added to the lysates along with antibodies against GluA1 N-terminal (Alomone labs, cat. #AGC-004).

Techniques: RNA Sequencing Assay, Immunostaining, Expressing, Western Blot, Transfection, Staining, Control, Plasmid Preparation, Knockdown, Two Tailed Test

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Western blot analysis revealed HHEX protein expression levels in brain lysates from WT and Ube3a 2xTg mice. ( B ) A notable decrease in HHEX was observed in Ube3a 2xTg mouse brains. n = 3 animals/group. ( C ) GluA1 RNA FISH (red) was conducted on rat primary cortical neurons that were transfected with either GFP, GFP + UBE3A, or GFP + UBE3A + HHEX. Scale bars, 10 μm. ( D ) UBE3A transfection resulted in nuclear retention of GluA1 mRNA, which was not rescued by HHEX co-transfection. GFP: n = 16, GFP + UBE3A: n = 10, GFP + UBE3A + HHEX: n = 11. Data information: Mean ± SEM. * p < 0.05; ** p < 0.01; ns = not significant. In ( B ), unpaired two-tailed t test. In ( D ), two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Protein A-Agarose beads (Santa Cruz Biotechnology; cat. #sc-2001) were added to the lysates along with antibodies against GluA1 N-terminal (Alomone labs, cat. #AGC-004).

Techniques: Western Blot, Expressing, Transfection, Cotransfection, Two Tailed Test

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Primary rat neurons were infected with either GFP virus, UBE3A virus, or both UBE3A and SARNP viruses. Neurons were treated with DMSO or puromycin for 12 h at DIV 11. After that, GluA1 was immunoprecipitated and probed for puromycin to detect GluA1 peptides being synthesized. Cell lysates (input) were also probed to detect total protein levels. ( B ) Quantification of puromycin-labeled GluA1 showed a decrease in GluA1 protein synthesis with UBE3A overexpression, which was reversed by SARNP restoration. n = 4 independent experiments. ( C ) Quantification of GluA1 abundance in input lysates. UBE3A overexpression caused a decrease in GluA1 abundance in total lysates, which was rescued by SARNP restoration. n = 4 independent experiments. ( D ) DIV10 rat primary neurons were transfected with GFP, GFP + UBE3A, or GFP + UBE3A + SARNP. GluA1 (red) and PSD95 (green) were immunostained to measure somatic and synaptic expression of GluA1 at DIV14. Scale bars, 25 μm (full picture); 10 μm (enlarged area). ( E – G ) SARNP restoration rescued the density of GluA1 puncta ( E ) and colocalized GluA1 + PSD95 puncta ( F ) along the dendrites, and GluA1 intensity in the soma ( G ). GFP: n = 16, GFP + UBE3A: n = 16, GFP + UBE3A + SARNP: n = 16. Data information: Mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant. One-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Protein A-Agarose beads (Santa Cruz Biotechnology; cat. #sc-2001) were added to the lysates along with antibodies against GluA1 N-terminal (Alomone labs, cat. #AGC-004).

Techniques: Infection, Virus, Immunoprecipitation, Synthesized, Labeling, Over Expression, Transfection, Expressing

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) c-Fos expression in rat primary neurons infected with GFP virus, UBE3A virus, or both UBE3A and SARNP viruses. Neurons were treated with either DMSO or 10 µM Glutamate (Glu) in the presence of APV + TTX or APV + TTX + CNQX. Hoechst was used to stain the nucleus. Scale bars,100 μm. ( B ) Quantification of the percentage of c-Fos positive cells. UBE3A overexpression abolished the Glu-induced increase in c-Fos positive neurons, which was rescued by SARNP restoration. GFP-TTX + APV: n = 13, GFP-Glu+TTX + APV: n = 13, GFP-Glu+TTX + APV + CNQX: n = 13, GFP + UBE3A-TTX + APV: n = 14, GFP + UBE3A-Glu+TTX + APV: n = 12, GFP + UBE3A-Glu+TTX + APV + CNQX: n = 13, GFP + UBE3A + SARNP-TTX + APV: n = 12, GFP + UBE3A + SARNP-Glu+TTX + APV: n = 14, GFP + UBE3A + SARNP-Glu+TTX + APV + CNQX: n = 14 images of 2.25 mm 2 area. ( C ) Representative traces of AMPAR mEPSCs obtained from whole-cell recordings at a holding potential of −70 mV. ( D , E, F ) Plots of mean amplitude ( D ), frequency ( E ) and Decay Tau ( F ) of mEPSCs recorded from DIV14-17 primary cortical neurons infected with GFP or UBE3A virus, together with SARNP or GluA1 virus for potential rescue. UBE3A overexpression impaired the amplitude of AMPAR mEPSCs, which was rescued by either SARNP or GluA1 restoration. GFP: n = 7, UBE3A: n = 9, UBE3A + SARNP: n = 8, UBE3A+GluA1: n = 8. Data information: Mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant. In ( B ), two-way ANOVA with Bonferroni’s multiple comparisons test. In ( D , E, F ), one-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Protein A-Agarose beads (Santa Cruz Biotechnology; cat. #sc-2001) were added to the lysates along with antibodies against GluA1 N-terminal (Alomone labs, cat. #AGC-004).

Techniques: Expressing, Infection, Virus, Staining, Over Expression