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Fig. 7. Podoplanin-induced EMT is associated with upregulation of RhoA activity. (A) Rho, Rac and Cdc42 bound to GTP affinity pull-down assays were used to determine the levels of active Rho GTPases. Levels of active RhoA, Rac1 and Cdc42 in MDCK and HaCaT cell clones transfected with the empty vector (EGFP) and with wild-type and mutant podoplanin constructs fused to EGFP. Quantification of RhoA-GTP expression level relative to total RhoA level was performed by densitometric analysis. Values below blots are relative to control (EGFP) cells, to which an arbitrary value of 1 was given. Results are representative of two experiments. (B) Western blot analysis of ERM phosphorylation relative to the total expression levels of ezrin and moesin. The levels of 
-tubulin were determined as a control for protein loading. (C) Western blot analysis of phospho-ERM levels relative to the total expression levels of ezrin and moesin in MDCK cells transfected with the empty vector (EGFP) and PWT before and after treatment with the Rock inhibitor Y27632. (D) Inhibition of RhoA signaling blocks podoplanin-stimulated cell migration. In vitro wound-healing assays of control (EGFP) and PWT-MDCK cells were performed (as in Fig. 5A) in the absence or presence of either Y27632 (which inhibits Rock) or soluble C3 transferase (which inhibits RhoA, RhoB and RhoC). Y27632 and C3 transferase reduced PWT-MDCK cell migration to basal and below basal levels, respectively. *P<0.05; ***P<0.001 vs PWT-MDCK cells.
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