Figure 3.
Figure 3. 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.

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.

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