Figure 5.

TGF-β treatment activates RhoA in epithelial cells. NMuMG cells cultured in serum-free medium were incubated with 10 ng/ml TGF-β for 0–15 min at 37°C in the presence or absence of 10 μg/ml LY294002 (LY) added 1 h before TGF-β treatment. (A) GTP loaded RhoA was measured by adsorbing cell lysates to GST-RBD beads and immunoblotted for RhoA. The densitometry measurements were normalized to total RhoA content of nonadsorbed cell lysate and represented by the bar graph (n = 4 ± SD). As a positive control, NMuMG cells treated for 5 min with LPA were assayed. TGF-β induces the rapid accumulation of RhoA-GTP in a PI3-kinase independent manner. (B) Further controls to show the GTP-loading potential of QL-RhoA– and N19-RhoA–transduced NMuMG cells assayed for RhoA activity. (C) NIH-3T3, primary keratinocytes (Pker), R1B, Mv1Lu, and BxPc3 cells were similarly assayed for RhoA activation. Only epithelial cells with intact TGF-β type I receptor exhibited TGF-β–mediated RhoA-GTP accumulation.