p190RhoGAP participates in the inhibition of RhoA by TGF-β1 signaling. (A) Cells were transfected with cDNA constructs encoding WT p190RhoGAP or DN p190RhoGAP lacking the GTP-binding domain or not transfected. After 36 hours, they were incubated with or without 5 ng/mL TGF-β1 for the indicated times. GTP-RhoA was determined by GST-RBD pull down, and total RhoA and HA-tagged p190RhoGAP were determined by Western blotting using anti-RhoA and anti-HA antibodies. The values are mean ± SE of 3 (None and DN) and 2 (WT) independent experiments (*P < .05). (B) Ninety percent confluent Raw264.7 cells in 6-well dishes (Corning) were transfected with 4 μg WT and DN p190RhoGAP constructs using the lipofectamine 2000 transfection reagent. After incubated for 24 hours, the cells were treated with 5 ng/mL TGF-β1 for 1 hour and 24 hours and then allowed to migrate in transwell cell culture chambers. Control and 5 ng/mL TGF-β1 pretreated cells were added to the upper reservoir. One milliliter of DMEM-F12 with chemotaxis reagents (5 ng/mL TGF-β1) was added in the lower reservoir. After 6 hours of migration, nonmigrated cells in the upper membrane were scraped off, and transmigrated cells in the lower membrane were stained by Giemsa staining method and then counted under microscopy. The values are means ± SE of 3 independent experiments, and each value was compared with corresponding control at the same time scale (**P < .01; ***P < .001).