notch1 icd  (Proteintech)

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation of keratinocytes and angiogenesis in diabetic mice. (a) Expression levels of integrin β1, K14, K10, K1, and Notch1 NICD evaluated by Western blot in the middle and at the end of wound healing. (b-f) Corresponding quantitative analysis. (g) CD31 and VEGF expression levels evaluated by Western blot. (h and i) Corresponding quantitative analysis. n = 3 in each group. ✶ P < 0.05. NICD: Notch Intracellular Domain, VEGF: Vascular endothelial growth factor, K14: Keratin 14, K10: Keratin 10, K1: Keratin 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation through interaction between HIF-1α and NICD. (a) Immunofluorescence showing the expression and co-localization of HIF-1α and NICD in HaCaT cells under different conditions; bar = 100 μm. (b) Western blot showing the expression levels of HIF-1α, NICD, K14, and integrin-β1 in HaCaT cells under different conditions. (c-f) Quantitative analysis of WB. n = 3 in each group; ✶ P < 0.05. (g) Co-IP confirming the interaction between HIF-1α and NICD. HIF-1: Hypoxia-inducible factor 1, NICD: Notch intracellular domain, WB: Western blot, Co-IP: Co-immunoprecipitation, K14: Keratin 14.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Immunofluorescence, Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Schematic model of roxadustat reversing the “high differentiated and low proliferation” state of keratinocytes in diabetic wounds. In normal skin, keratinocytes maintain a relatively stable rhythm of proliferation and differentiation. Injury can activate the process of wound repair, upregulate HIF-1 signal, downregulate Notch1 signal, and make keratinocytes in a state of “high proliferation and low differentiation.” In the context of diabetes, the skin is usually thin and wound healing is delayed, HIF-1 signaling is inhibited, and Notch1 signaling is continuously activated, making keratinocytes in a state of “low proliferation and high differentiation.” FG-4592 upregulates the inhibited HIF-1 signaling and downregulates the hyperactivated Notch1 signaling, which both benefit diabetic wound re-epithelialization. By Fig draw (version 2.0, www.figdraw.com ). HIF-1: Hypoxia-inducible factor 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Western blot of hdBEC lysates cultured under static or flow (∼20 dynes/cm ) conditions, with pre-treatment with 0.5 μg/mL of rhDll4 or vehicle control. (B) Quantification of normalized Western blot intensity of Notch1 V1754. (C) Western blot of hdBEC lysates from scramble (SCR), DLL4 KO , and JAG1 KO hdBECs under static and flow conditions. (D) Quantification of normalized Western blot intensity of Notch1 V1754. (E) Western blot of hdBEC and hdLEC lysates under static and flow conditions. (F) Quantification of normalized Western blot intensity of Notch1 V1754. (G) Fluorescence micrographs of hdBECs and hdLECs under static and flow conditions immunostained for Notch1 ICD (heatmap) and VE-cadherin (white). Scale bar, 25 μm. (H) Quantification of the relative Notch1 polarization in hdBECs versus hdLECs under flow. n ≥ 10 fields of view from three independent experiments. (I) Timelapse of Notch1-GFP in hdBEC cells under flow. Time scale (min:sec). Scale bar, 10 μm. (J) Fluorescence micrographs of SCR and DLL4 KO cells under flow conditions immunostained for Notch1 (black) and DNA (blue). Scale bar, 25 μm. (K) Quantification of the relative degree of Notch1 polarization in SCR versus DLL4 KO cells under flow. n ≥ 10 fields of view from three independent experiments. (K) Quantification of the relative degree of ligand polarization in Dll4-GFP or Jag1-mEmerald cells under flow. n ≥ 10 fields of view from three independent experiments. Western blots are representative of three independent experiments. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Western Blot, Cell Culture, Control, Fluorescence

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Left: Fluorescence micrographs of co-culture of hdBECs expressing mEmerald (green) or mApple (magenta) under flow. Scale bar, 5 μm. Inset: High magnification micrograph of the cell-cell interface with mApple, mEmerald, and Notch1 ECD (cyan). Yellow inset: XZ orthogonal projection. Scale bar, 5 μm. Right: experimental schematic. (B) Fluorescence micrographs of hdBECs pretreated for 1 h with 10 μM DAPT, 1 μM BB94 or DMSO vehicle control and cultured under flow. Scale bar, 25 μm. (C) Quantification of Notch1 polarization in hdBECs treated with DAPT, BB94, or DMSO vehicle control under flow. n ≥ 10 fields of view from three independent experiments. (D) Super-resolution by optical pixel reassignment fluorescence micrographs of the downstream cell-cell interface of flow-polarized hdBECs. Scale bar, 5 μm. (E) Line scan quantification of relative Notch1 ECD (magenta) and Notch1 ICD (green) distribution (representative yellow dashed line). n = 10 profiles. (F) Fluorescence time series from live cell movie of hdBECs labeled with AF647-Notch1 ECD antibody under flow. Pseudo-colored to depict cell-cell boundaries. Insets (i) and (ii) of distinct polarized domains where individual particles (red circles) are tracked over time moving retrograde opposite the direction of flow. Time scale (min:sec). Scale bar, 5 μm. (G) Schematic illustrating pulse-chase Notch1 ECD antibody labeling experiments. (H) Fluorescence micrographs of internalized Notch1 in SCR and DLL4 KO cells under flow conditions from pulse-chase labeling of Notch1 ECD (black). Scale bar, 25 μm. (I) Quantification of endocytosed Notch1 in SCR versus DLL4 KO cells. n ≥ 10 fields of view from three independent experiments. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Fluorescence, Co-Culture Assay, Expressing, Control, Cell Culture, Labeling, Pulse Chase, Antibody Labeling

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Wild-type Notch1 (WT) or a Notch1 mutant lacking the intracellular domain (ΔICD) C-terminally tagged with mEmerald. (B) Western blots of Notch1-WT expressing hdBEC lysates cultured under static and flow conditions or plated on control or rhDll4-coated substrate. Quantification: Notch1 V1754 intensity normalized to total construct expression (mEmerald recognized by GFP antibody). (C) Western blot of Notch1-ΔICD expressing hdBEC lysates cultured under static and flow conditions or plated on rhDll4-coated dishes. Quantification: Notch1 V1754 intensity normalized to total construct expression (mEmerald recognized by GFP antibody). Quantification of Western blot intensity of Notch1 V1754 normalized to GFP. n = 3 independent experiments. (D) Western blot of lysates from Notch1-ΔICD expressing hdBEC monolayers mosaically co-cultured with increasing percentage of Dll4-mScarlet overexpressing hdBECs. Quantification is Notch1 V1754 intensity normalized to GFP. (E) Fluorescence micrographs of Notch1-WT and Notch1-ΔICD expressing hdBECs under flow. Yellow arrows denote flow-polarized domains. Scale bar, 25 μm. (F) Quantification of Notch1 polarization in Notch1-WT and Notch1-ΔICD expressing hdBECs under flow. n ≥ 10 fields of view from three independent experiments. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, **p < 0.01, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Mutagenesis, Western Blot, Expressing, Cell Culture, Control, Construct, Fluorescence

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Select list of ICD-interacting proteins identified via mass spectrometry to increase under flow. (B) Western blot of Notch1 co-immunoprecipitation from hdBECs cultured under static or flow conditions. (C) Western blot of lysates from SCR and ANXA2 KO hdBECs under flow. (D) Quantification of Western blot intensity of Notch1 V1754 normalized to GAPDH. Data is normalized to SCR for n = 3 independent experiments. (E) Fluorescence micrograph of annexin A2 in hdBECs under flow. Scale bar, 25 μm. (F) Fluorescence micrographs of endocytosis of Notch1 from pulse-chase labelled of SCR and ANXA2 KO cells. Purple dashed lines indicate cell segmentation. Scale bar, 25 μm. (G) Quantification of internalized Notch1 ECD in SCR versus ANXA2 KO cells. n ≥ 10 fields of view from three independent experiments. (H) Associated quantification of the relative degree of Notch1 polarization in SCR versus ANXA2 KO cells under flow, measured as described previously. n ≥ 10 fields of view from three independent experiments. (I) Fluorescence micrographs of caveolin-1 and VE-cadherin in hdBECs under flow. Scale bar, 25 μm. (J) Western blot of hdBEC lysates from SCR and CAV1 KO cells cultured under static and flow conditions or plated on rhDll4-coated substrates. (K) Quantification of Western blot intensity of Notch1 V1754 normalized to GAPDH. Data is normalized to SCR static condition. (L) Quantification of internalized Notch1 ECD by pulse-chase labeling in SCR versus CAV1 KO cells. n ≥ 10 fields of view from three independent experiments. (M) Quantification of Notch1 polarization in SCR versus CAV1 KO cells under flow. n ≥ 10 fields of view from three independent experiments. (N) Western blot of detergent-resistant membranes (DRM) fractions isolated from SCR and CAV1 KO hdBEC cells under static and flow conditions. (O) Fluorescence micrographs of hdBECs and hdLECs under flow. Scale bar, 25 μm. (P) Schematic depicting flow-polarized downstream domains where (a) ICD localizes full-length Notch1, (b) annexin A2 regulates Notch1 cis-endocytosis, (c) and caveolin-1 establishes microdomains compartmentalizing Notch1 and γ-secretase. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Mass Spectrometry, Western Blot, Immunoprecipitation, Cell Culture, Fluorescence, Pulse Chase, Labeling, Isolation

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: Following primary antibodies were used: chicken anti-MAP2 (1:5000, Millipore Sigma, Cat#AB5543, RRID: AB_571049), mouse anti-Jag1 (1:1000, Santa Cruz Biotechnology, Cat# sc-390177, RRID: AB_2892141), goat anti-Jag2 (1:1000, Thermo Fisher Scientific, Cat# PA5–47188, RRID: AB_2576459), rabbit anti-Jag2 (1:1000, Cell Signaling Technology, Cat# 2210, RRID: AB_823553), goat anti-Dll1 (1:1000, Abcam, Cat# ab85346, RRID: AB_1860332), rabbit anti-Dll1 (1:1000, Cell Signaling Technology, Cat#2588, RRID: AB_2292961), goat anti-Dll4 (1:1000, Thermo Fisher Scientific, Cat# PA5–46974, RRID: AB_2577158), rabbit anti-Dll4 (1:1000, Cell Signaling Technology, Cat# 96406, RRID: AB_2800263), guinea pig anti-VGluT1 (1:1000, Millipore Sigma, Cat# AB5905, RRID: AB_2301751), rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730), mouse anti-Tsg101 (1:1000, Santa Cruz Biotechnology, Cat# sc-7964, RRID: AB_671392), rabbit anti-Notch1 ICD (1:1000, Abcam, Cat# ab52627, RRID: AB_881725), mouse anti-LBPA (1:500, Millipore Sigma, Cat # MABT837), rabbit anti-Notch2 ICD (1:1000, Cell Signaling Technology, Cat# 5732S, RRID: AB_10693319), mouse anti-PSD95 (1:500, Thermo Fisher Scientific, Cat # MA1–046, RRID: AB_2092361), chicken anti-GFP (1:5000, Abcam, Cat# ab13970, RRID: AB_300798) and mouse anti-myc (1:1000, Santa Cruz Biotechnology, Cat# sc-40, RRID: AB_627268).

Techniques: Purification, Negative Staining, Isolation, Tandem Mass Spectroscopy, 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: Following primary antibodies were used: chicken anti-MAP2 (1:5000, Millipore Sigma, Cat#AB5543, RRID: AB_571049), mouse anti-Jag1 (1:1000, Santa Cruz Biotechnology, Cat# sc-390177, RRID: AB_2892141), goat anti-Jag2 (1:1000, Thermo Fisher Scientific, Cat# PA5–47188, RRID: AB_2576459), rabbit anti-Jag2 (1:1000, Cell Signaling Technology, Cat# 2210, RRID: AB_823553), goat anti-Dll1 (1:1000, Abcam, Cat# ab85346, RRID: AB_1860332), rabbit anti-Dll1 (1:1000, Cell Signaling Technology, Cat#2588, RRID: AB_2292961), goat anti-Dll4 (1:1000, Thermo Fisher Scientific, Cat# PA5–46974, RRID: AB_2577158), rabbit anti-Dll4 (1:1000, Cell Signaling Technology, Cat# 96406, RRID: AB_2800263), guinea pig anti-VGluT1 (1:1000, Millipore Sigma, Cat# AB5905, RRID: AB_2301751), rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730), mouse anti-Tsg101 (1:1000, Santa Cruz Biotechnology, Cat# sc-7964, RRID: AB_671392), rabbit anti-Notch1 ICD (1:1000, Abcam, Cat# ab52627, RRID: AB_881725), mouse anti-LBPA (1:500, Millipore Sigma, Cat # MABT837), rabbit anti-Notch2 ICD (1:1000, Cell Signaling Technology, Cat# 5732S, RRID: AB_10693319), mouse anti-PSD95 (1:500, Thermo Fisher Scientific, Cat # MA1–046, RRID: AB_2092361), chicken anti-GFP (1:5000, Abcam, Cat# ab13970, RRID: AB_300798) and mouse anti-myc (1:1000, Santa Cruz Biotechnology, Cat# sc-40, RRID: AB_627268).

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: Following primary antibodies were used: chicken anti-MAP2 (1:5000, Millipore Sigma, Cat#AB5543, RRID: AB_571049), mouse anti-Jag1 (1:1000, Santa Cruz Biotechnology, Cat# sc-390177, RRID: AB_2892141), goat anti-Jag2 (1:1000, Thermo Fisher Scientific, Cat# PA5–47188, RRID: AB_2576459), rabbit anti-Jag2 (1:1000, Cell Signaling Technology, Cat# 2210, RRID: AB_823553), goat anti-Dll1 (1:1000, Abcam, Cat# ab85346, RRID: AB_1860332), rabbit anti-Dll1 (1:1000, Cell Signaling Technology, Cat#2588, RRID: AB_2292961), goat anti-Dll4 (1:1000, Thermo Fisher Scientific, Cat# PA5–46974, RRID: AB_2577158), rabbit anti-Dll4 (1:1000, Cell Signaling Technology, Cat# 96406, RRID: AB_2800263), guinea pig anti-VGluT1 (1:1000, Millipore Sigma, Cat# AB5905, RRID: AB_2301751), rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730), mouse anti-Tsg101 (1:1000, Santa Cruz Biotechnology, Cat# sc-7964, RRID: AB_671392), rabbit anti-Notch1 ICD (1:1000, Abcam, Cat# ab52627, RRID: AB_881725), mouse anti-LBPA (1:500, Millipore Sigma, Cat # MABT837), rabbit anti-Notch2 ICD (1:1000, Cell Signaling Technology, Cat# 5732S, RRID: AB_10693319), mouse anti-PSD95 (1:500, Thermo Fisher Scientific, Cat # MA1–046, RRID: AB_2092361), chicken anti-GFP (1:5000, Abcam, Cat# ab13970, RRID: AB_300798) and mouse anti-myc (1:1000, Santa Cruz Biotechnology, Cat# sc-40, RRID: AB_627268).

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: Following primary antibodies were used: chicken anti-MAP2 (1:5000, Millipore Sigma, Cat#AB5543, RRID: AB_571049), mouse anti-Jag1 (1:1000, Santa Cruz Biotechnology, Cat# sc-390177, RRID: AB_2892141), goat anti-Jag2 (1:1000, Thermo Fisher Scientific, Cat# PA5–47188, RRID: AB_2576459), rabbit anti-Jag2 (1:1000, Cell Signaling Technology, Cat# 2210, RRID: AB_823553), goat anti-Dll1 (1:1000, Abcam, Cat# ab85346, RRID: AB_1860332), rabbit anti-Dll1 (1:1000, Cell Signaling Technology, Cat#2588, RRID: AB_2292961), goat anti-Dll4 (1:1000, Thermo Fisher Scientific, Cat# PA5–46974, RRID: AB_2577158), rabbit anti-Dll4 (1:1000, Cell Signaling Technology, Cat# 96406, RRID: AB_2800263), guinea pig anti-VGluT1 (1:1000, Millipore Sigma, Cat# AB5905, RRID: AB_2301751), rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730), mouse anti-Tsg101 (1:1000, Santa Cruz Biotechnology, Cat# sc-7964, RRID: AB_671392), rabbit anti-Notch1 ICD (1:1000, Abcam, Cat# ab52627, RRID: AB_881725), mouse anti-LBPA (1:500, Millipore Sigma, Cat # MABT837), rabbit anti-Notch2 ICD (1:1000, Cell Signaling Technology, Cat# 5732S, RRID: AB_10693319), mouse anti-PSD95 (1:500, Thermo Fisher Scientific, Cat # MA1–046, RRID: AB_2092361), chicken anti-GFP (1:5000, Abcam, Cat# ab13970, RRID: AB_300798) and mouse anti-myc (1:1000, Santa Cruz Biotechnology, Cat# sc-40, RRID: AB_627268).

Techniques: One-tailed Test, Staining, 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: Following primary antibodies were used: chicken anti-MAP2 (1:5000, Millipore Sigma, Cat#AB5543, RRID: AB_571049), mouse anti-Jag1 (1:1000, Santa Cruz Biotechnology, Cat# sc-390177, RRID: AB_2892141), goat anti-Jag2 (1:1000, Thermo Fisher Scientific, Cat# PA5–47188, RRID: AB_2576459), rabbit anti-Jag2 (1:1000, Cell Signaling Technology, Cat# 2210, RRID: AB_823553), goat anti-Dll1 (1:1000, Abcam, Cat# ab85346, RRID: AB_1860332), rabbit anti-Dll1 (1:1000, Cell Signaling Technology, Cat#2588, RRID: AB_2292961), goat anti-Dll4 (1:1000, Thermo Fisher Scientific, Cat# PA5–46974, RRID: AB_2577158), rabbit anti-Dll4 (1:1000, Cell Signaling Technology, Cat# 96406, RRID: AB_2800263), guinea pig anti-VGluT1 (1:1000, Millipore Sigma, Cat# AB5905, RRID: AB_2301751), rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730), mouse anti-Tsg101 (1:1000, Santa Cruz Biotechnology, Cat# sc-7964, RRID: AB_671392), rabbit anti-Notch1 ICD (1:1000, Abcam, Cat# ab52627, RRID: AB_881725), mouse anti-LBPA (1:500, Millipore Sigma, Cat # MABT837), rabbit anti-Notch2 ICD (1:1000, Cell Signaling Technology, Cat# 5732S, RRID: AB_10693319), mouse anti-PSD95 (1:500, Thermo Fisher Scientific, Cat # MA1–046, RRID: AB_2092361), chicken anti-GFP (1:5000, Abcam, Cat# ab13970, RRID: AB_300798) and mouse anti-myc (1:1000, Santa Cruz Biotechnology, Cat# sc-40, RRID: AB_627268).

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: Following primary antibodies were used: chicken anti-MAP2 (1:5000, Millipore Sigma, Cat#AB5543, RRID: AB_571049), mouse anti-Jag1 (1:1000, Santa Cruz Biotechnology, Cat# sc-390177, RRID: AB_2892141), goat anti-Jag2 (1:1000, Thermo Fisher Scientific, Cat# PA5–47188, RRID: AB_2576459), rabbit anti-Jag2 (1:1000, Cell Signaling Technology, Cat# 2210, RRID: AB_823553), goat anti-Dll1 (1:1000, Abcam, Cat# ab85346, RRID: AB_1860332), rabbit anti-Dll1 (1:1000, Cell Signaling Technology, Cat#2588, RRID: AB_2292961), goat anti-Dll4 (1:1000, Thermo Fisher Scientific, Cat# PA5–46974, RRID: AB_2577158), rabbit anti-Dll4 (1:1000, Cell Signaling Technology, Cat# 96406, RRID: AB_2800263), guinea pig anti-VGluT1 (1:1000, Millipore Sigma, Cat# AB5905, RRID: AB_2301751), rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730), mouse anti-Tsg101 (1:1000, Santa Cruz Biotechnology, Cat# sc-7964, RRID: AB_671392), rabbit anti-Notch1 ICD (1:1000, Abcam, Cat# ab52627, RRID: AB_881725), mouse anti-LBPA (1:500, Millipore Sigma, Cat # MABT837), rabbit anti-Notch2 ICD (1:1000, Cell Signaling Technology, Cat# 5732S, RRID: AB_10693319), mouse anti-PSD95 (1:500, Thermo Fisher Scientific, Cat # MA1–046, RRID: AB_2092361), chicken anti-GFP (1:5000, Abcam, Cat# ab13970, RRID: AB_300798) and mouse anti-myc (1:1000, Santa Cruz Biotechnology, Cat# sc-40, RRID: AB_627268).

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: Following primary antibodies were used: chicken anti-MAP2 (1:5000, Millipore Sigma, Cat#AB5543, RRID: AB_571049), mouse anti-Jag1 (1:1000, Santa Cruz Biotechnology, Cat# sc-390177, RRID: AB_2892141), goat anti-Jag2 (1:1000, Thermo Fisher Scientific, Cat# PA5–47188, RRID: AB_2576459), rabbit anti-Jag2 (1:1000, Cell Signaling Technology, Cat# 2210, RRID: AB_823553), goat anti-Dll1 (1:1000, Abcam, Cat# ab85346, RRID: AB_1860332), rabbit anti-Dll1 (1:1000, Cell Signaling Technology, Cat#2588, RRID: AB_2292961), goat anti-Dll4 (1:1000, Thermo Fisher Scientific, Cat# PA5–46974, RRID: AB_2577158), rabbit anti-Dll4 (1:1000, Cell Signaling Technology, Cat# 96406, RRID: AB_2800263), guinea pig anti-VGluT1 (1:1000, Millipore Sigma, Cat# AB5905, RRID: AB_2301751), rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730), mouse anti-Tsg101 (1:1000, Santa Cruz Biotechnology, Cat# sc-7964, RRID: AB_671392), rabbit anti-Notch1 ICD (1:1000, Abcam, Cat# ab52627, RRID: AB_881725), mouse anti-LBPA (1:500, Millipore Sigma, Cat # MABT837), rabbit anti-Notch2 ICD (1:1000, Cell Signaling Technology, Cat# 5732S, RRID: AB_10693319), mouse anti-PSD95 (1:500, Thermo Fisher Scientific, Cat # MA1–046, RRID: AB_2092361), chicken anti-GFP (1:5000, Abcam, Cat# ab13970, RRID: AB_300798) and mouse anti-myc (1:1000, Santa Cruz Biotechnology, Cat# sc-40, RRID: AB_627268).

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