Wnt3 adependent Cell Motility Involves Rho A Activation
- Slides: 14
Wnt-3 a-dependent Cell Motility Involves Rho. A Activation and Is Specifically Regulated by Dishevelled-2 J. Biol. Chem. , Vol. 280, Issue 1, 777 -786, January 7, 2005
Cell line CHO Hamster Chinese ovary, Epithelial Migration assay Wound Healing Assay transwell assay
Wnt-3 a induced morphological changes in CHO cells Phalloidin staining
Wnt-3 a CM-stimulated migration of CHO cells. Wound Healing Assay (24 hr) transwell assay (6 hr)
b -Catenin pathway was stimulated by Wnt-3 a CM but not required for migration in transwell assay.
Rho. A was activated by Wnt-3 a CM and contributed to Wnt-3 a-dependent cell migration.
Wnt-3 a induced phosphorylation of Dvl-2 and Dvl-3
Intracellular distribution of Dvl-2 following treatment with Wnt-3 a or L CM. Wnt 3 a CM L CM
Knock-down of endogenous Dvl-2 by RNAi inhibited Wnt-3 a-induced cell motility.
GST-RBD pull-down assay the pull-down assay used to detect active small GTPases the N-terminal region of Raf 1 protein kinase contains the Ras–binding domain (RBD) the N-terminal regulatory region in p 21 -activated protein kinase 1 and 2 (Pak 1 and 2) contains the p 21 -binding domain (PBD) for Rac and Cdc 42, the N-terminal region of Rhotekin contains the Rho-binding domain (RBD). The GTPase-binding domains from these downstream effectors are expressed as recombinant glutathione S-transferase (GST) fusion proteins immobilized on glutath and can be used to affinity precipitate (pull-down) the active GTPase from cell lysates. Pulled-down active GTPases are eluted from the resin and detected by immunoblotting with a specifi