1. J. Biol. Chem., Vol. 280, Issue 1, 777-786, January 7, 2005
Wnt-3a-dependent Cell Motility Involves RhoA Activation and Is Specifically Regulated by Dishevelled-2
J. Biol. Chem., Vol. 280, Issue 1, , January 7, 2005

2. Migration assay Cell line CHO Hamster Chinese ovary, Epithelial
Wound Healing Assay
transwell assay

3. Wnt-3a induced morphological changes in CHO cells
Phalloidin staining

4. Wnt-3a CM-stimulated migration of CHO cells.
transwell assay (6hr)
Wound Healing Assay (24hr)

5. b -Catenin pathway was stimulated by Wnt-3a CM
but not required for migration in transwell assay.

7. RhoA was activated by Wnt-3a CM
and contributed to Wnt-3a-dependent cell migration.

8. Wnt-3a induced phosphorylation of Dvl-2 and Dvl-3

9. Intracellular distribution of Dvl-2 following treatment with Wnt-3a or L CM.
Wnt3a CM
L CM

10. Knock-down of endogenous Dvl-2 by RNAi
inhibited Wnt-3a-induced cell motility.

14. GST-RBD pull-down assay
the pull-down assay used to detect active small GTPases
the N-terminal region of Raf1 protein kinase contains the Ras–binding domain (RBD)
the N-terminal regulatory region in p21-activated protein kinase 1 and 2 (Pak1 and 2)
contains the p21-binding domain (PBD) for Rac and Cdc42,
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 glutathione resin
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 specific antibody