Journal: Scientific Reports

Article Title: Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

doi: 10.1038/s41598-020-69454-5

Figure Lengend Snippet: Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p < 0.05 relative to control, t-test).

Article Snippet: Neurons were stained in non-permeabilized conditions with primary antibodies against extracellular epitopes of GluN2A, GluN2B or GluA1 (ACG-002 1:500, Alomone Labs , , ACG-003 1:500, Alomone Labs , , and PC246 1:1000, MERCK ) and then they were depleted of cholesterol.

Techniques: Marker

Journal: Nature Communications

Article Title: Elevated synaptic PKA activity and abnormal striatal dopamine signaling in Akap11 mutant mice, a genetic model of schizophrenia and bipolar disorder

doi: 10.1038/s41467-025-66504-2

Figure Lengend Snippet: a Schematic of AKAP11 immunoprecipitation (IP) and its most notable, known interactors. b Immunoblot of AKAP11 in IPs of anti-AKAP11 and control IgG in 12-week-old (12wk) WT or Akap11 -/- cortical tissue. This experiment was repeated in >5 independent experiments, with similar results. c Volcano plot comparing Log Fold-Change (Log FC) against P value for AKAP11 IP in WT vs Akap11 -/- , displaying all proteins detected by coIP-MS. A moderated two-sample t-test was applied to the sample group datasets. Proteins that were enriched (red) by anti-AKAP11 IP from WT versus Akap11 -/- cortical extracts. Enriched proteins, defined by having a nominal P value < 0.05 and Log FC > 0, are colored red. Downregulated proteins are mostly antibody-related artifacts and can be found in Supplementary Data . d Venn diagram comparing previously published AKAP11 interactions with the brain interactome in this study. Full results are displayed in Supplementary Data . e Immunoblot of AKAP11, PKA subunits and control proteins, in IPs of AKAP11, GluA1 or IgG control in WT or Akap11 -/- cortical lysate. This experiment was repeated in >3 independent experiments, with similar results. f Immunoblot of AKAP11 in IPs of PKA subunits, in WT cortical lysates. This experiment was repeated in >2 independent experiments, with similar results. g–j Immunoblot with indicated antibodies, in IPs of anti-GSK3α, GSK3β, VAPA, VAPB, AKAP11, P62 or DYRK1a in WT or Akap11 -/- cortical lysate. These experiments were repeated in at least 2 independent experiments, with similar results. k Gene set enrichment analysis of all proteins displayed in 1c, showing selected gene ontologies with a positive normalized enrichment score (NES) and adjusted P -value (FDR) < 0.01. Redundant (similar) pathways were removed for clarity. The top most enriched proteins within each ontology are listed within the histograms. GSEA uses a Kolmogorov-Smirnov test, two tailed, with Benjamini-Hochberg (B-H) False Discovery Rate (FDR) correction applied.

Article Snippet: For immunoblot, the following antibodies were used: 1:1000 Rabbit anti-AKAP11 (Custom R171), 1:1000 Rabbit anti-AKAP11 (Custom R173), 1:1000 Mouse anti-PKA R1α (Thermo Fisher Scientific MA5-24981), 1:1000 Rabbit anti-PKA R1α (CST 5675), 1:1000 Rabbit anti-PKA Cα (CST 4782), 1:1000 Rabbit anti-Iqgap1 (Abcam ab86064), 1:1000 Rabbit anti-Dyrk1a (CST 2771), 1:1000 Rabbit anti-GSK3α (CST 4818), 1:1000 Rabbit anti-GSK3β (CST 9315), 1:1000 Mouse anti-P62 (Abcam ab56416), 1:1000 Rabbit anti-LC3A/B (CST 12741), 1:1000 Rabbit anti-VAPA (Proteintech 15275-1-AP), 1:1000 Rabbit anti-β Catenin (CST 8480), 1:1000 Mouse anti-Protein Phosphatase 1 (Santa Cruz SC-7482), 1:1000 Rabbit anti-Sik2 (CST 6919), 1:1000 Rabbit anti-p-GSK3α S21 / anti-p-GSK3β S9 (CST 8566), 1:1000 Rabbit anti-p-GSK3α / anti-p-GSK3β (CST 5676), 1:1000 Rabbit anti- p-GSK3β S9 (CST 9336), 1:1000 Rabbit anti- p-GluA1 S845 (CST 8084), 1:1000 Rabbit anti- GluA1 (CST 13185), 1:1000 Mouse anti- GluA1 (NeuroMab 75-327), 1:1000 Mouse anti-PSD95 (BioLegend 810301), 1:1000 Rabbit anti-Homer1 (Synaptic Systems 160003), 1:1000 Guinea Pig anti-Synaptophysin 1(Synaptic Systems 101004).

Techniques: Immunoprecipitation, Western Blot, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Elevated synaptic PKA activity and abnormal striatal dopamine signaling in Akap11 mutant mice, a genetic model of schizophrenia and bipolar disorder

doi: 10.1038/s41467-025-66504-2

Figure Lengend Snippet: a Number of phosphoproteins reaching a significance threshold of nominal P < 0.05. Fractions of upregulated and downregulated DAPPs are highlighted in red and blue, respectively. b Volcano plots of 12wk synapse phosphoproteomics data for Akap11 +/- and Akap11 -/- vs. WT controls. Phosphoproteomic measurements are normalized to MS-proteomics measurements from the same samples. Known PKA Cα substrates from PhosphoSitePlus are labeled green. AKAP11 phosphopeptides are omitted from Akap11 -/- plots, for clarity. a,b A moderated two-sample t-test was applied to the datasets to compare WT, Akap11 +/- and Akap11 -/- sample groups. c Motif analysis of peptides flanking the phosphorylation site of P < 0.05 phosphosites in A. n(fg) are the number of foreground peptides, and n(bg) are the number of background peptides. Red lines indicate FDR significance, calculated by log odds enrichment. d PTM-SEA of synapse phosphoproteomics data, using known kinase substrates from PhosphoSitePlus. Black borders indicate FDR significance. e ELISA-based PKA activity measurements comparing total lysate and synapse fractions of cortex (left) and striatum-enriched tissue (right). One Unit (U) is defined by the manufacturer as the quantity of PKA that catalyzes the transfer of 1.0 pmol phosphate from ATP to substrate. Two-way ANOVA with Tukey’s post hoc test. f Immunoblotting and quantification of AKAP11, PKA subunits, p-GluA1 S845 and GluA1 in 12wk cortical total lysate. One-way ANOVA with Tukey’s post hoc test. e,f Data are represented as mean ± SEM. See Supplementary Data for detailed statistical information.

Article Snippet: For immunoblot, the following antibodies were used: 1:1000 Rabbit anti-AKAP11 (Custom R171), 1:1000 Rabbit anti-AKAP11 (Custom R173), 1:1000 Mouse anti-PKA R1α (Thermo Fisher Scientific MA5-24981), 1:1000 Rabbit anti-PKA R1α (CST 5675), 1:1000 Rabbit anti-PKA Cα (CST 4782), 1:1000 Rabbit anti-Iqgap1 (Abcam ab86064), 1:1000 Rabbit anti-Dyrk1a (CST 2771), 1:1000 Rabbit anti-GSK3α (CST 4818), 1:1000 Rabbit anti-GSK3β (CST 9315), 1:1000 Mouse anti-P62 (Abcam ab56416), 1:1000 Rabbit anti-LC3A/B (CST 12741), 1:1000 Rabbit anti-VAPA (Proteintech 15275-1-AP), 1:1000 Rabbit anti-β Catenin (CST 8480), 1:1000 Mouse anti-Protein Phosphatase 1 (Santa Cruz SC-7482), 1:1000 Rabbit anti-Sik2 (CST 6919), 1:1000 Rabbit anti-p-GSK3α S21 / anti-p-GSK3β S9 (CST 8566), 1:1000 Rabbit anti-p-GSK3α / anti-p-GSK3β (CST 5676), 1:1000 Rabbit anti- p-GSK3β S9 (CST 9336), 1:1000 Rabbit anti- p-GluA1 S845 (CST 8084), 1:1000 Rabbit anti- GluA1 (CST 13185), 1:1000 Mouse anti- GluA1 (NeuroMab 75-327), 1:1000 Mouse anti-PSD95 (BioLegend 810301), 1:1000 Rabbit anti-Homer1 (Synaptic Systems 160003), 1:1000 Guinea Pig anti-Synaptophysin 1(Synaptic Systems 101004).

Techniques: Phospho-proteomics, Labeling, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot

Journal: Nature chemical biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: A) Heat map analysis shows the PCR-based microarray analysis of plasticity-associated genes in the hippocampus of WT and αKO ( Ppara -null) mice. Three mice were used in each group. B) Venn diagram of plasticity-associated genes shows the number of genes inhibited (28; red circle), stimulated (34; green circle) and unchanged (22; overlapped region) in Ppara -null hippocampus. C) Real-time PCR analyses of Arc, Creb, Grin2a, Grin2b, and Gria1 mRNAs were performed to confirm the array results. Results are mean ± SEM of three mice. a p<0.001 vs WT . D) Hippocampal tissue of 6- to 8-week-old WT (n=3) and Ppara -null (n=3) mice were immunostained for MAP-2 (green) and PSD-95 (red). The representative image was taken from CA1 region of the hippocampus. Scale bar = 10μm. E) The magnified view of region enclosed in the box is shown in the image. Scale bar = 10 μm. Results represent analysis of three hippocampal sections of each of three mice per group. The expression of NR-2A, GluR1, PSD95, Arc, and CREB in hippocampal tissue of WT ( n=3 ) and Ppara -null ( n=3 ) mice was further assessed by Western blot (F) followed by densitometric analyses (G) after normalizing with actin. For raw uncut blots, please see . Results are mean ± SEM of three mice. a p<0.001 vs WT .

Article Snippet: Rabbit polyclonal anti-PPARα antibody (Abcam; Cat# ab189159; WB and IHC), mouse anti-NeuN antibody (Millipore; Cat# MAB377), rabbit polyclonal anti-PPARβ antibody (Abcam; Cat # ab8937; WB and IHC), anti-PPARγ antibody (Abcam; Cat# ab66343; WB and IHC), anti-NMDAR2A antibody (Cell Signaling for WB at a dilution of 1:1000, Cat #4205; Abcam for IHC, Cat# ab169873), anti-GluR1 antibody (Cell Signaling for WB at a dilution of 1:1000, Cat #13185; Abcam for IHC, Cat # ab131507), anti-CREB antibody ( Cell Signaling for WB at a dilution of 1:1000 and IC at a dilution of 1:200, Cat# 9104), and anti-Arc antibody (Abcam for WB at a dilution of 1:1000, Cat # ab118929) were used in this study.

Techniques: Microarray, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Molecular medicine reports

Article Title: Naringin ameliorates memory deficits and exerts neuroprotective effects in a mouse model of Alzheimer's disease by regulating multiple metabolic pathways.

doi: 10.3892/mmr.2021.11971

Figure Lengend Snippet: Figure 8. Naringin exerts neuroprotective effects by regulating the glutamate receptor system and apoptosis. (A) Protein levels of NMDAR1, GluR2 and CAMKII. (B) Protein levels of Bad, Bcl‑2 and cleaved‑caspase‑3. **P<0.01 vs. sham group; #P<0.05 and ##P<0.01 vs. model group; &P<0.05, &&P<0.01 vs. naringin group. NMDAR1, glutamate receptor subunit 1; GluR2, glutamate receptor 2; CAMKII, calcium/calmodulin‑dependent protein kinase type II.

Article Snippet: After blocking with 5% non‐fat dried milk for 2.5 h at 37 ̊C, the PVDF membrane was incubated with primary antibodies against: APP (1:1,000; cat. no. bs‐12503R; BIOSS), BACE1 (1:1,000; cat. no. 5606T; Cell Signaling Technology, Inc.), CDK5 (1:1,000; cat. no. bs‐10258Rm; BIOSS), p‐Tau396 (1:1,000; cat. no. bs‐3446R; BIOSS), glutamate receptor subunit 1 (NMDAR1) (1:1,000; cat. no. bs‐23343R; BIOSS), glutamate receptor 2 (GluR2; 1:1,000; cat. no. bs‐13385R; BIOSS), calcium/calmod‐ ulin‐dependent protein kinase type II (CAMKII; 1:1,000; Boster), Bad (1:500; cat. no. A00183; Boster), caspase‐3 (1:500; cat. no. bs‐0081R; BIOSS), Bcl‐2 (1:500; cat. no. bs‐0032R; BIOSS), ERβ (1:1,000; cat. no. kl437Hu22; KALANG; https://www.biomart.cn/infosupply/31407572.htm), p‐P38 (1:1,000; cat. no. bs‐2210R; BIOSS) and β‐actin (1:1,000; cat. no. bs‐0061R; BIOSS) overnight.

Techniques: