1 / 14

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, 777-786, January 7, 2005. Cell line. Hamster Chinese ovary, Epithelial. CHO. Migration assay. Wound Healing Assay. transwell assay.

duy
Download Presentation

J. Biol. Chem., Vol. 280, Issue 1, 777-786, January 7, 2005

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

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

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

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

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

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

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

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

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

  10. 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

More Related