Cultured Cells
Cultured cells are cells extracted and cultured from humans and mice, and are mainly used to analyze intracellular molecular mechanisms such as gene expression levels and protein-protein interactions. In this laboratory, we use cultured cells to analyze the molecular mechanisms by which Notch signaling is activated.
1. molecular mechanism of Notch signaling activation regulated by Mib
It has been shown that Mib1 ubiquitinates Delta and Jagged in ligand-expressing cells and activates Notch signaling in adjacent Notch-expressing cells, but the detailed mechanism has not been clarified. In this laboratory, we are analyzing the contribution of Mib1 focusing on (1) ligand binding to the Notch receptor, (2) uptake of the Notch extracellular domain, and (3) ligand recycling.
2) Elucidation of the regulatory mechanism of Notch signaling activity using ligand proteins
In this laboratory, purified ligand proteins are added to Notch-expressing cells and stimulated, and Notch signaling is measured over time using a system in which luciferase is expressed in response to Notch signaling activation. Using this method, we are searching for novel proteins that contribute to the activity of Notch signaling and analyzing the domains of Delta and Jagged that are required for Notch signal activation.
The cultured cell group performs these analyses using Western Blotting, immunoprecipitation, cell immunostaining and microscopy, measurement of Notch signal activity (Luciferase assay), and measurement of transcriptional activity using real-time PCR.