antibodies against notch1 v1754  (Cell Signaling Technology Inc)

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: 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: Biomedical Reports

Article Title: Notch signaling pathway regulates the progression of fetal growth restriction through mediating immune dysfunction

doi: 10.3892/br.2025.1989

Figure Lengend Snippet: The expression and distribution of Notch1 and Jagged1 is decreased in PBMCs and placenta. (A-E) The expression of Notch1 and Jagged1 in PBMCs and placenta was detected by (A and B) reverse transcription-quantitative PCR, (C) western blotting, (D) immunofluorescence and (E) immunohistochemistry, respectively. ** P<0.01 and *** P<0.001 vs. control. Scale bar, 100 µm. PBMCs, peripheral blood mononuclear cells; FGR, fetal growth restriction.

Article Snippet: After antigen retrieval, the samples were incubated overnight at 4 ̊C with primary antibodies against Notch1 (1:200), Jagged1 (1:200), CD3 (1:200; cat. no. 17617-1-AP), CD86 (1:200; cat. no. 26903-1-AP), CD206 (1:200; cat. no. 18704-1-AP; last 3 obtained from Proteintech Group, Inc.) and Forkhead Box protein 3 (Foxp3; 1:200; cat. no. ab36607; Abcam).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Immunohistochemistry, Control