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Fig. 3. Smad7-dependent activation of the Rho GTPases. (A) PC-3U/pMEP4-S7 cells were serum-starved in 1% FBS for 12 hours and treated with 1 µM CdCl2 for 12 hours or with 1 µM CdCl2 together with 5 ng/ml TGF-ß for 15 minutes up to 48 hours. The control cells, PC-3U, were stimulated with 5 ng/ml of TGF-ß for 12 hours as positive control, and with CdCl2 for 12 hours as negative control. The amount of active, GTP-loaded Rac1, Cdc42 and RhoA was determined by GST pull-down assays with GST-PAK-CRIB, GST-WASP-CRIB or GST-Rhotekin, respectively. Rac1, Cdc42 and RhoA were detected by immunoblotting using antibodies specific for the respective GTPase. (B) Immunoblots were analyzed by densitometry and the data were combined into diagrams showing the activation of Rac1, Cdc42 and RhoA. Each column represents the mean + s.e.m. of three independent experiments.





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