Journal: The Journal of Neuroscience

Article Title: Neurogenic Subventricular Zone Stem/Progenitor Cells Are Notch1-Dependent in Their Active But Not Quiescent State

doi: 10.1523/JNEUROSCI.0455-12.2012

Figure Lengend Snippet: Primary and secondary antibodies used in the analysis

Article Snippet: Antibodies used in the experiments are described in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antigen Species Dilution, 1: Source Notch1 (intracellular domain) Rabbit 1000 Nyfeler et al., 2005 Notch1 (extracelluar domain) Rat 100 H. R. MacDonald Notch2 (extracelluar domain) Rat 100 H. R. MacDonald Notch3 Goat 100 R&D Systems bGal Rabbit 2000 CAPPEL PCNA Mouse 1000 Dako North America Doublecortin Guinea pig 2000 Chemicon Sox2 Rabbit 500 Chemicon BrdU Rat 2000 Serotec Ascl1 (Mash1) Mouse 200 F. Guillemot GFAP Mouse 500 Sigma-Aldrich Neuronal nuclear antigen (NeuN) Mouse 500 Sigma FITC/Cy3/Cy5 conjugated anti-mouse, rabbit, rat, goat Donkey 1000 Jackson Immunoresearch Open in a separate window Primary and secondary antibodies used in the analysis X-Gal staining and quantification.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neurogenic Subventricular Zone Stem/Progenitor Cells Are Notch1-Dependent in Their Active But Not Quiescent State

doi: 10.1523/JNEUROSCI.0455-12.2012

Figure Lengend Snippet: Notch2 and Notch3 are expressed in the SVZ with a similar pattern to Notch1. A, Notch1 is expressed in the SVZ of adult mice by GFAP+ and GFAP− cells. B, C, Consecutive sections stained with anti-Notch2 (B) or anti-Notch3 (C) antibodies showing a comparable expression pattern in the SVZ to that of Notch1. D, Notch2 is expressed by GFAP− cells in the subependymal region of the SVZ but also by GFAP+ SVZ astrocytes and putative NSCs in close proximity to or making contact with the lateral ventricle. E, Some Notch2+GFAP− cells are Doublecortin+ (Dcx+) neuroblasts. F, G, Notch3 is prominently expressed by GFAP+ SVZ astrocytes. Scale bars: A–C, 100 μm; D–G, 20 μm.

Article Snippet: Antibodies used in the experiments are described in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antigen Species Dilution, 1: Source Notch1 (intracellular domain) Rabbit 1000 Nyfeler et al., 2005 Notch1 (extracelluar domain) Rat 100 H. R. MacDonald Notch2 (extracelluar domain) Rat 100 H. R. MacDonald Notch3 Goat 100 R&D Systems bGal Rabbit 2000 CAPPEL PCNA Mouse 1000 Dako North America Doublecortin Guinea pig 2000 Chemicon Sox2 Rabbit 500 Chemicon BrdU Rat 2000 Serotec Ascl1 (Mash1) Mouse 200 F. Guillemot GFAP Mouse 500 Sigma-Aldrich Neuronal nuclear antigen (NeuN) Mouse 500 Sigma FITC/Cy3/Cy5 conjugated anti-mouse, rabbit, rat, goat Donkey 1000 Jackson Immunoresearch Open in a separate window Primary and secondary antibodies used in the analysis X-Gal staining and quantification.

Techniques: Staining, Expressing

Journal: Cell reports

Article Title: Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication

doi: 10.1016/j.celrep.2024.113680

Figure Lengend Snippet: (A) Experimental scheme depicting EV purification workflow and cargo analyses. (B) Representative images of EV particles present in SEC fraction 2 (F2) visualized by negative staining EM. Scale bar, 500 nm; inset scale bar, 50 nm. (C) The diameter range of EV-like particles. n = 299 particles from two biological replicates. (D) The EV markers Alix, Tsg101, and CD81 but not GM130 are selectively detected in SEC F2 isolated from glycine-stimulated neuronal cultures. (E) Proteomic analysis reveals that Notch1 and Notch2 are highly abundant in SEC F2 along with other known EV protein markers. Mean ± SEM from 3 biological replicates. Insert: Notch1 and Notch2 peptides identified by MS/MS. (F) WB validation of Notch1 ICD and Notch2 ICD in SEC F2 (top). Silver-stained gel indicating the total amount of protein loaded across the 10 fractions (bottom). See also and .

Article Snippet: Rabbit anti-activated Notch2 , MyBioSource , Cat# MBS9410510.

Techniques: Purification, Negative Staining, Isolation, Tandem Mass Spectroscopy, Biomarker Discovery, Staining

Journal: Cell reports

Article Title: Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication

doi: 10.1016/j.celrep.2024.113680

Figure Lengend Snippet: (A) Proteinase K (PK) treatment reduces the apparent Notch1 and Notch2 molecular weight by ~10 kDa, consistent with removal of the extracellular portion of Notch ICD . No intact CD81 was detected in the PK-treated EVs presumably due to multiple cleavage events, while Sdcbp was unaffected by PK treatment. (B) Schematic illustrating the arrangement of Notch1, Notch2, CD81, and Sdcbp in EVs. (C) Representative ICC images showing Notch ligands Jag1, Jag2, Dll1, and Dll4 colocalize with internalized neuronal EVs, labeled with CFSE. Top, neuronal soma, scale bar, 10 μm; bottom, dendrites, scale bar, 5 μm (D) Quantification of the percentage of CFSE-labeled EVs colocalized with the indicated Notch ligands in neuronal soma. One-tailed Student’s t test, n = 12–15 from two cultures. NS, not significant. *p value (Jag1 vs. Jag2) = 0.0204, **p value (Jag1 vs. Dll1) = 0.0082, **p value (Jag1 vs. Dll4) = 0.0051, p value (Jag2 vs. Dll1) = 0.2947, p value (Jag2 vs. Dll4) = 0.2476, p value (Dll1 vs. Dll4) = 0.4396. (E) Left, CFSE labeled EVs that are internalized by primary cultured rat hippocampal neurons remain generally intact and punctate. Right, neurons fail to internalize PK-treated EVs. Scale bar, 10 μm. (F) Following a 60-min incubation of primary cultured rat hippocampal neurons with EVs, there is a notable increase in the levels of activated Notch1, activated Notch2, and Hes1 proteins. This effect is not observed with EVs treated with Delta MAX or with Delta MAX treatment alone. (G–I) Quantifications of (F). One-tailed Student’s t test, n = 4 biological. (G) ***p value (Veh vs. EV) = 0.0002, ***p value (EV vs. Delta MAX -treated EV) = 0.0004, NS p value (Delta MAX -treated EV vs. Delta MAX ) = 0.4453. (H) **p value (Veh vs. EV) = 0.0043, **p value (EV vs. Delta MAX -treated EV) = 0.0042, NS p value (Delta MAX -treated EV vs. Delta MAX ) = 0.4927. (I) ***p value (Veh vs. EV) = 0.0004, **p value (EV vs. Delta MAX -treated EV) = 0.0011, NS p value (Delta MAX -treated EV vs. Delta MAX ) = 0.2677. See also and .

Article Snippet: Rabbit anti-activated Notch2 , MyBioSource , Cat# MBS9410510.

Techniques: Molecular Weight, Labeling, One-tailed Test, Cell Culture, Incubation

Journal: Cell reports

Article Title: Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication

doi: 10.1016/j.celrep.2024.113680

Figure Lengend Snippet: (A) Top, experimental scheme showing the analysis time points for bulk neuron RNA analysis. Bottom, biological replicates cluster by analysis time point in multidimensional scaling plots. (B) Heatmap showing a panel of Notch target genes that are activated by Mg 2+ -free glycine treatment. (C) Volcano plot depicting comparison of mRNA levels in Veh-treated neurons (5 min after treatment) and Mg 2+ -free glycine-treated neurons (60 min after treatment). One-tailed Student’s t test. (D) Representative WB blot showing Mg 2+ -free glycine treatment also elevated the levels of activated Notch1, activated Notch2, and Hes1 in primary cultured rat hippocampal neurons (90 min after treatment), which can be inhibited by either D-2-amino-5-phosphonovalerate (APV) or dynasore. (E) Quantification of (D). n = 4 biological replicates. One-tailed Student’s t test. Hes1: **p value (Veh vs. Gly) = 0.0065, *p value (Gly vs. +APV) = 0.0102, **p value (+DMSO vs. +dynasore) = 0.0076. Activated Notch1: **p value (Veh vs. Gly) = 0.0003, **p value (Gly vs. +APV) = 0.0008, *p value (+DMSO vs. +dynasore) = 0.0102. Activated Notch2: ***p value (Veh vs. Gly) = 7.466E-05, ***p value (Gly vs. +APV) = 5.215E-05, **p value (+DMSO vs. +dynasore) = 0.0015. See also .

Article Snippet: Rabbit anti-activated Notch2 , MyBioSource , Cat# MBS9410510.

Techniques: Comparison, One-tailed Test, Cell Culture

Journal: Cell reports

Article Title: Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication

doi: 10.1016/j.celrep.2024.113680

Figure Lengend Snippet: (A) Top, representative fluorescent detection of Mg 2+ -free glycine-induced EVs from neurons with indicated genotypes. Scale bar, 10 μm. Bottom, quantification of Mg 2+ -free glycine-induced neuronal EVs immunocaptured from indicated neuron genotypes. n = 3 biological replicates (BR). Each chip contains three technical replicates. One-tailed Student’s t test. ***p value ( Alix +/+ vs. Alix +/− ) = 7.821-E07, ***p value ( Alix +/+ vs. Alix −/− ) = 3.143-E15, **p value ( Alix +/− vs. Alix −/− ) = 0.0060. (B) Left, WB analysis showing Mg 2+ -free glycine stimulation failed to induce EV release in Alix −/− hippocampal neurons. Right, silver-stained gel indicating the total amount of protein recovered across the 10 size exclusion fractions. (C) WB analysis showing Mg 2+ -free glycine stimulation failed to upregulate the level of activated Notch1, activated Notch2, and Hes1in Alix −/− hippocampal neurons. (D) Quantification of (C). n = 3 biological replicates. One-tailed Student’s t test. NS, not significant. Activated Notch1: p value = 0.3598. Activated Notch2: p value = 0.2162. Hes1: p value = 0.2737. (E) WB analysis showing Mg 2+ -free glycine stimulation elevated the levels of activated Notch1, activated Notch2, and Hes1in Alix +/+ but not Alix −/− hippocampal neurons from littermates. (F) Quantification of (E). n = 4 cultures each genotype. One-tailed Student’s t test. Activated-Notch1: *p value = 0.0128. Activated Notch2: ***p value = 0.0007. Hes1, ***p value = 0.0004. (G) Mg 2+ -free glycine stimulation leads to Alix phosphorylation, which can be inhibited by PKA inhibitor H89. p-S/T, phosphorylated serine or threonine. Stau, staurosporine. (H) Quantification of (G). n = 4 biological replicates. One-tailed Student’s t test. NS, not significant. ***p value (Veh+DMSO vs. Gly+DMSO) = 1.050E-07, p value (Veh+DMSO vs. Gly+H89) = 0.1624, ***p value (Gly+DMSO vs. Gly+H89) = 2.443E-07, p value (Gly+DMSO vs. Gly+Stau) = 0.3850. (I) Representative MS 2 spectra indicating Alix phosphorylation at S717 from rat hippocampal neuron whole-cell extracts treated with Mg 2+ -free glycine. Assigned fragment ions are indicated in b (blue) and y (red), and those containing phosphorylated serine 717 are labeled. (J) AlphaFold 2-predicted 3D protein structures of rat Alix, indicating the position of S717. (K) Overexpression of mCherry-Alix or mCherry-Alix-S717D rescues Mg 2+ -free glycine-induced EV release from Alix −/− neurons. Scale bar, 10 μm. (L) Quantification of (K). n = 4–7 biological replicates. Each chip contains three technical replicates. One-tailed Student’s t test. NS, not significant. ***p value (mCherry vs. mCherry-Alix) = 4.249E-06, p value (mCherry vs. mCherry-Alix-S717A) = 0.2110, ***p value (mCherry vs. mCherry-Alix-S717D) = 6.004E-13, ***p value (mCherry-Alix vs. mCherry-Alix-S717A) = 5.468E-10, p value (mCherry-Alix vs. mCherry-Alix-S717D) = 0.1312. See also and and .

Article Snippet: Rabbit anti-activated Notch2 , MyBioSource , Cat# MBS9410510.

Techniques: One-tailed Test, Staining, Phospho-proteomics, Labeling, Over Expression

Journal: Cell reports

Article Title: Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication

doi: 10.1016/j.celrep.2024.113680

Figure Lengend Snippet: (A) WB analyses showing that, at P0 and P4, the levels of activated Notch1, activated Notch2, Notch1 ICD , Notch2 ICD , and Hes1 are similar in Alix +/+ and Alix −/− hippocampi. However, at P14, the levels of activated Notch1, activated Notch2, and Hes1 are significantly reduced in Alix −/− hippocampus. (B) Quantification of (A). n = 3 mice for each group. One-tailed Student’s t test, NS, not significant. P0: activated Notch1 p value = 0.3752, activated Notch2 p value = 0.3865, Notch1 ICD p value = 0.2047, Notch2 ICD p value = 0.3590, Hes1 p value = 0.1232. P4: activated Notch1 p value = 0.2149, activated Notch2 p value = 0.2743, Notch1 ICD p value = 0.3837, Notch2 ICD p value = 0.4283, Hes1 p value = 0.0836. P14: activated Notch1 *p value = 0.0324, activated Notch2 *p value = 0.0265, Notch1 ICD p value = 0.1029, Notch2 ICD p value = 0.4227, Hes1 **p value = 0.0042. (C) At P0 and P4, the expression patterns of Notch1 ICD and Notch2 ICD are similar in Alix +/+ and Alix −/− hippocampal CA1 regions. At P14, the amounts of nuclear-localized Notch1 ICD and Notch2 ICD are much less in Alix −/− hippocampal CA1 regions compared to Alix +/+ hippocampus. Scale bar, 10 μm. See also .

Article Snippet: Rabbit anti-activated Notch2 , MyBioSource , Cat# MBS9410510.

Techniques: One-tailed Test, Expressing

Journal: Cell reports

Article Title: Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication

doi: 10.1016/j.celrep.2024.113680

Figure Lengend Snippet: (A) The levels of activated Notch1, activated Notch2, and Hes1 are significantly reduced in hippocampus from ~2-month-old Camk2a-cre :: Alix fl/fl mice compared to Alix fl/fl mice. M = mouse. (B) Quantification of (A). n = 4 mouse per group. One-tailed Student’s t test. NS, not significant. Activated Notch1: *p value = 0.0143, activated Notch2: **p value = 0.0075, Notch1 ICD : p value = 0.0942, Notch2 ICD : p value = 0.4709, Hes1: *p value = 0.0218, Alix: ***p value = 5.346E-05. (C) Lack of Alix in adult hippocampus led to alteration of nuclear-localized Notch1 ICD and Notch2 ICD . Scale bar, 10 μm (D) Quantification of (C). n = 5 mouse per group. One-tailed Student’s t test. NS, not significant. Nucleic Notch1 ICD /cytosolic Notch1 ICD : CA1, **p value = 0.0042, CA3, **p value = 0.0037, DG, ***p value = 0.0002. Nucleic Notch2 ICD /cytosolic Notch2 ICD : CA1, **p value = 0.0026, CA3, ***p value = 0.0009, DG, p value = 0.3942.

Article Snippet: Rabbit anti-activated Notch2 , MyBioSource , Cat# MBS9410510.

Techniques: One-tailed Test

Journal: Cell reports

Article Title: Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication

doi: 10.1016/j.celrep.2024.113680

Figure Lengend Snippet:

Article Snippet: Rabbit anti-activated Notch2 , MyBioSource , Cat# MBS9410510.

Techniques: Virus, Recombinant, Protease Inhibitor, Lactate Dehydrogenase Assay, Magnetic Beads, Silver Staining, Phospho-proteomics, Peptide Fractionation, RNA Sequencing, Mass Spectrometry, Software, Sequencing, Transmission Assay, Imaging, Microscopy