sheep anti human n3ecd  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not alter the steady-state levels of the NOTCH3 WT or C185R protein. A , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells with and without doxycycline-inducible expression of RFNG. NOTCH3 and RFNG were detected by antibodies against human NOTCH3 extracellular domain (ECD) and human RFNG. TUBULIN was blotted with an antibody against human/mouse α-tubulin as a loading control. The white and black arrowheads indicate full-length NOTCH3 and the ECD, respectively. B , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells treated with or without 0, 0.1, 0.5, or 1 μg/ml doxycycline. RFNG was detected by an antibody against HA, which tagged RFNG. The black arrowheads indicate N3ECDs. C , relative amounts of N3ECD WT and C185R with or without RFNG expression, as determined by Western blotting (B) (independent biological replicates N = 3). N3ECD band intensities were normalized to those observed without doxycycline. D , representative Western blot of cell lysates collected from cultured N3WT-RF-HeLa and N3C185R-RF-HeLa cells with or without RFNG expression and treated with puromycin for 0 or 16 h. The black arrowheads indicate N3ECDs. E , relative band intensities of N3ECD at 16 h normalized to those observed at 0 h, as determined by Western blotting in (D) (independent biological replicates N = 3). Data information: In C and E , data are presented as mean ± SD. n.s., not significant (C: Tukey’s post hoc test following one-way ANOVA; E: Tukey’s post hoc test following two-way ANOVA). HA, hemagglutinin; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Western Blot, Cell Culture, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG enhances JAG1-induced degradation of NOTCH3 WT but not that of R141C and C185R. A , images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/C185R-RF-HeLa with MIG-3T3 or JAG1-3T3 cells. N3WT/C185R-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The left panel shows GFP expressed in MIG-3T3 and JAG1-3T3 ( green ). The middle panel shows N3ECD ( magenta ). The right panel shows an overlay. Arrows indicate N3ECD transendocytosed into JAG1-3T3. The scale bar represents 10μm. B , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and C185R (independent biological replicates, N = 4). C , representative images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/R141C-RF-HeLa with JAG1-3T3 cells. N3WT/R141C-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The scale bar represents 10μm. D , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and R141C (independent biological replicates, N = 5). Data information: In B and D , data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, n.s., not significant (Tukey’s post hoc test following three-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not affect JAG1-mediated transendocytosis of NOTCH3 WT and C185R. A , schematic representation of the immunocytochemistry workflow used to separately detect NOTCH3 present in the cell membrane and in JAG1-3T3 cells. After N3WT/C185R-RF-HeLa cells transfected with NOTCH2 siRNA were cocultured with JAG1-3T3 cells, the fixed cells were stained with two different anti-NOTCH3ECD (N3ECD) antibodies (clones 1G5 and BAF1559) before and after cell permeabilization, respectively. The scale bars indicate 20 μm. B , representative confocal images of cocultured N3WT/C185R-RF-HeLa and JAG1-3T3 cells, as depicted in (A). The white arrowheads indicate NOTCH3 particles detected by antibodies against N3ECD (clones 1G5 and BAF1559), indicating the presence of NOTCH3 on the cell membrane. The scale bars indicate 20 μm. C , the ratio of NOTCH3 present on the cell membrane against the total NOTCH3 in JAG1-3T3 cells. Box plots: 10 to 90 percentile. (independent biological replicates: N = 3; total cell numbers, WT RFNG-: n = 86, WT RFNG+: n = 86, C185R RFNG-: n = 93, C185R RFNG+: n = 81). ∗∗p < 0.01, ∗∗∗∗ p < 0.0001 (Tukey’s post hoc tests following two-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Membrane, Transfection, Staining, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not alter the steady-state levels of the NOTCH3 WT or C185R protein. A , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells with and without doxycycline-inducible expression of RFNG. NOTCH3 and RFNG were detected by antibodies against human NOTCH3 extracellular domain (ECD) and human RFNG. TUBULIN was blotted with an antibody against human/mouse α-tubulin as a loading control. The white and black arrowheads indicate full-length NOTCH3 and the ECD, respectively. B , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells treated with or without 0, 0.1, 0.5, or 1 μg/ml doxycycline. RFNG was detected by an antibody against HA, which tagged RFNG. The black arrowheads indicate N3ECDs. C , relative amounts of N3ECD WT and C185R with or without RFNG expression, as determined by Western blotting (B) (independent biological replicates N = 3). N3ECD band intensities were normalized to those observed without doxycycline. D , representative Western blot of cell lysates collected from cultured N3WT-RF-HeLa and N3C185R-RF-HeLa cells with or without RFNG expression and treated with puromycin for 0 or 16 h. The black arrowheads indicate N3ECDs. E , relative band intensities of N3ECD at 16 h normalized to those observed at 0 h, as determined by Western blotting in (D) (independent biological replicates N = 3). Data information: In C and E , data are presented as mean ± SD. n.s., not significant (C: Tukey’s post hoc test following one-way ANOVA; E: Tukey’s post hoc test following two-way ANOVA). HA, hemagglutinin; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Western Blot, Cell Culture, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG enhances JAG1-induced degradation of NOTCH3 WT but not that of R141C and C185R. A , images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/C185R-RF-HeLa with MIG-3T3 or JAG1-3T3 cells. N3WT/C185R-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The left panel shows GFP expressed in MIG-3T3 and JAG1-3T3 ( green ). The middle panel shows N3ECD ( magenta ). The right panel shows an overlay. Arrows indicate N3ECD transendocytosed into JAG1-3T3. The scale bar represents 10μm. B , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and C185R (independent biological replicates, N = 4). C , representative images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/R141C-RF-HeLa with JAG1-3T3 cells. N3WT/R141C-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The scale bar represents 10μm. D , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and R141C (independent biological replicates, N = 5). Data information: In B and D , data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, n.s., not significant (Tukey’s post hoc test following three-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not affect JAG1-mediated transendocytosis of NOTCH3 WT and C185R. A , schematic representation of the immunocytochemistry workflow used to separately detect NOTCH3 present in the cell membrane and in JAG1-3T3 cells. After N3WT/C185R-RF-HeLa cells transfected with NOTCH2 siRNA were cocultured with JAG1-3T3 cells, the fixed cells were stained with two different anti-NOTCH3ECD (N3ECD) antibodies (clones 1G5 and BAF1559) before and after cell permeabilization, respectively. The scale bars indicate 20 μm. B , representative confocal images of cocultured N3WT/C185R-RF-HeLa and JAG1-3T3 cells, as depicted in (A). The white arrowheads indicate NOTCH3 particles detected by antibodies against N3ECD (clones 1G5 and BAF1559), indicating the presence of NOTCH3 on the cell membrane. The scale bars indicate 20 μm. C , the ratio of NOTCH3 present on the cell membrane against the total NOTCH3 in JAG1-3T3 cells. Box plots: 10 to 90 percentile. (independent biological replicates: N = 3; total cell numbers, WT RFNG-: n = 86, WT RFNG+: n = 86, C185R RFNG-: n = 93, C185R RFNG+: n = 81). ∗∗p < 0.01, ∗∗∗∗ p < 0.0001 (Tukey’s post hoc tests following two-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Membrane, Transfection, Staining, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Determination of Blood NOTCH3 Extracellular Domain and Jagged-1 Levels in Healthy Subjects

doi: 10.3390/ijms231810547

Figure Lengend Snippet: NOTCH3 extracellular domain (N3ECD) levels in healthy adults. N3ECD levels were measured in serum and plasma from healthy adults by ELISA ( n = 279) ( A ) and a correlation between serum and plasma N3ECD levels was analyzed ( B ). Jagged-1 levels were measured in serum and plasma ( C ) and their correlation was assessed ( D ). Each symbol represents a value from a single subject; midline represents the mean; and error bars represent the standard error. The resulting Spearman’s correlation coefficient ( r ) and corresponding p value are reported.

Article Snippet: Standards and samples were loaded onto plates and incubated at RT for 2 h. Captured N3ECD was detected using biotinylated polyclonal antibody raised against the N3ECD (R&D Systems; BAF1559; 1000ng/mL).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Determination of Blood NOTCH3 Extracellular Domain and Jagged-1 Levels in Healthy Subjects

doi: 10.3390/ijms231810547

Figure Lengend Snippet: Correlations between N3ECD and Jag-1 levels. Positive correlations of serum ( A ) and plasma levels ( B ) between N3ECD and Jag1 ( n = 279). Results are representative of three independent ELISA experiments. The resulting Spearman’s correlation coefficient ( r ) and corresponding p -values are reported.

Article Snippet: Standards and samples were loaded onto plates and incubated at RT for 2 h. Captured N3ECD was detected using biotinylated polyclonal antibody raised against the N3ECD (R&D Systems; BAF1559; 1000ng/mL).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Determination of Blood NOTCH3 Extracellular Domain and Jagged-1 Levels in Healthy Subjects

doi: 10.3390/ijms231810547

Figure Lengend Snippet: Correlations between blood N3ECD levels and clinical characteristics.

Article Snippet: Standards and samples were loaded onto plates and incubated at RT for 2 h. Captured N3ECD was detected using biotinylated polyclonal antibody raised against the N3ECD (R&D Systems; BAF1559; 1000ng/mL).

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Determination of Blood NOTCH3 Extracellular Domain and Jagged-1 Levels in Healthy Subjects

doi: 10.3390/ijms231810547

Figure Lengend Snippet: Comparison of mean N3ECD and Jagged-1 levels by gender and age. Serum and plasma N3ECD levels between male and female ( A ) and correlations between N3ECD levels and age ( B ). Jagged-1 serum and plasma levels in male and female ( C ) and correlations with age ( D ). Each symbol represents values from a single subject; midline represents the mean; and error bars represent the standard error. The resulting Spearman’s correlation coefficient ( r ) and corresponding p -value are reported. ns—not significant.

Article Snippet: Standards and samples were loaded onto plates and incubated at RT for 2 h. Captured N3ECD was detected using biotinylated polyclonal antibody raised against the N3ECD (R&D Systems; BAF1559; 1000ng/mL).

Techniques: Comparison, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Determination of Blood NOTCH3 Extracellular Domain and Jagged-1 Levels in Healthy Subjects

doi: 10.3390/ijms231810547

Figure Lengend Snippet: Correlations between serum N3ECD levels and laboratory values. Correlations of N3ECD with creatinine ( A ), and hemoglobin ( B ). Correlations of serum Jag-1 with systolic blood pressure ( C ), HbA1c ( D ), and triglyceride ( E ). Each symbol represents values from a single subject. The resulting Spearman’s correlation coefficient ( r ) and corresponding p value are reported.

Article Snippet: Standards and samples were loaded onto plates and incubated at RT for 2 h. Captured N3ECD was detected using biotinylated polyclonal antibody raised against the N3ECD (R&D Systems; BAF1559; 1000ng/mL).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL

doi: 10.1084/jem.20161715

Figure Lengend Snippet: A13 Notch3 agonist antibody activates a ligand-insensitive Notch3 receptor with the C455R mutation in vitro. (A) Schematic diagram showing the structure of the Notch3 receptor and the binding site where the A13 antibody interacts with the NRR domain. Lin12-Notch (LNR), heterodimerization domain (HD), and transmembrane domain (TM) are shown. Luciferase reporter assay for Notch signaling in monocultures of HEK 293 cells expressing Notch3 WT receptor (B) or the Notch3 C455R (C) after treatment with A13 or control IgG antibodies. TET indicates concentration of tetracycline used to induce transgene expression. Luciferase reporter assay for Notch signaling in HEK 293 cell co-cultures. HEK 293 cells expressing Notch3 WT or C455R Notch3 receptors in co-culture with control cells (D) or with HEK 293 cells expressing Jagged 1 (E). HEK 293 cells expressing Notch3 WT in co-culture with control cells (F) or with cells expressing Jagged 1 (G) treated with IgG or A13 antibodies. HEK 293 cells expressing Notch3 C455R in co-culture with control cells (H) or with cells expressing Jagged 1 (I) treated with IgG or A13. ELISA measurements of N3ECD in cell culture supernatant for each culture, Notch3 WT (J) and C455R (K). Luciferase reporter assay for Notch signaling in monocultures of HEK 293 cells expressing Notch3 WT receptor in the presence of compound E (L). ELISA measurements of N3ECD in cell culture supernatant in the presence of compound E (M). Luciferase reporter assay for Notch signaling in monocultures of HEK cells expressing Notch3 WT receptor treated with increasing concentrations of A13 (N). ELISA measurements of N3ECD in cell culture supernatant from cells treated with increasing concentrations of A13 (O). Notch3 signaling activation measured via gene reporter assay correlated with increased levels of N3ECD in cell culture supernatants measured by ELISA (P). (B–P) *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were analyzed via ANOVA. Values in graphs are expressed as the means ± SEM. The results are representative of three independent experiments each including three technical replicates per condition.

Article Snippet: The recombinant human N3ECD, originally used to raise the antibodies (1559-NT-050; R&D Systems), was used as the standard, and a positive control.

Techniques: Mutagenesis, In Vitro, Binding Assay, Luciferase, Reporter Assay, Expressing, Control, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL

doi: 10.1084/jem.20161715

Figure Lengend Snippet: A13 Notch3 agonist antibody reverses plasma biomarker changes in N3ECD and endostatin/collagen 18α1 in CADASIL mice. Dot plots showing plasma levels of N3ECD (A), endostatin/collagen 18α1 (B), IGFBP-1 (C), and HTRA1 (D) in 6-wk-old C455R mice treated with the A13 ( n = 10) or IgG ( n = 8) antibodies from 10 independent rounds of injections. (A-D) **, P < 0.01. Statistical analysis was done via unpaired two-tailed Student's t test. Each data point represents the value for a single mouse. Horizontal lines show the means ± SEM. The C455R mice were littermates.

Article Snippet: The recombinant human N3ECD, originally used to raise the antibodies (1559-NT-050; R&D Systems), was used as the standard, and a positive control.

Techniques: Clinical Proteomics, Biomarker Discovery, Two Tailed Test