Fig. 4.
TβRIII-mediated Cdc42 activation, inhibition of migration, and alterations in the actin cytoskeleton are mediated through the interaction of TβRIII with β-arrestin2. (A) Transwell migration toward a 10% FBS gradient was assessed 48 h after infecting Ovca429 or Ovca433 cells with adenovirus-expressing GFP, full-length TβRIII (TβRIII), TβRIII unable to bind β-arrestin2 (TβRIII-T841A), or TβRIII unable to bind GIPC (TβRIII-DEL). Fold migration relative to GFP control is presented. Data represent the mean ± SE for 4 independent experiments for Ovca429 and 2 independent experiments for Ovca433, each done in triplicate. (#, P = 0.0189 and *, P = 0.0440). (B–D) The indicated cells were fixed and stained for actin using rhodamine phalloidin. GFP-expressing cells were examined and representative examples are presented. Scale bar = 6.6 μm. (E and G) Cells were serum starved and stimulated with TGF-β1 (200 pM) for the indicated times and pull-down assays for active Cdc42 conducted. Quantification is presented in Fig. S7C andFig. S7D, respectively. (F) Ovca429TβRIII cells were subjected to siRNA-mediated silencing of β-arrestin2 (Inset) and transwell migration performed toward 10% FBS. Fold migration relative to Neo controls are presented. Data represent the mean ± SE of 2 independent experiments.