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Previous knowledge: Asef2 promotes cell migration when cells are plated on…
Previous knowledge: Asef2 promotes cell migration when cells are plated on fibronectin
Asef2 inhibits cell migration on type I collagen
GFP–Asef2 transfection and Asef2 knockdown (HT1080 and MDA-MB-231)
Migration assay
(Rose plot of migration tracs)
Rac activity is enhanced by Asef2
Pulldown assay (Detection of active Rac)
FRET analysis
(Activity Rac detection)
Asef2 promotes larger adhesions that turn over slowly on type I collagen
Immunostaining for paxillin
(adhesion marker)
Adhesion turnover quantification (Half-life)
Flow cytometry
(HUTS-4 AB - binds to active b1 integrin)
TIRF microscopy
Traction force and cell contractility are enhanced by Asef2
Traction force microscopy;
Vector maps of traction stresses
Gel contraction assay
Asef2 regulates migration by increasing MyoII activity
Immunostaining, fluorescence microscopy
(AB against MyoII PO3ed at S19)
Immunostaining, fluorescence microscopy
(AB against Akt PO3 on Thr308)
Activation of Rac by Asef2 regulates MyoII contractility
Knockdown of Rac (shRNA) and
MyoII activity assessment
(AB against MyoII PO3ed at S19)
Treatment with NSC23766 (Rac inhibitor)
Gel contraction assay