FIG 6.
Tension activates Rho GEFs through p66Shc and FAK. For the assays who results are presented in panels A to G, cells were exposed to fibronectin-coated microbeads and a magnetic field was applied for 5 min. (A, B) Total cellular GEF activity of p115-RhoGEF (A) and GEF-H1 (B) obtained by pulldown with RhoA(G17A) is shown. GST-RhoA (wild type) was included as a negative control. (C to F) GEF activity for p115-RhoGEF (C, E) and GEF-H1 (D, F) is shown following knockdown of p66Shc (C, D) or FAK (E, F). (G) (Left) RhoA activation in response to tension is shown in cells transduced with control (con), GEF-H1, or p115-RhoGEF shRNA. (Center and right) Protein levels after knockdown. The knockdown efficiency was ∼69% (GEF-H1) and 92% (p115). (H) Cells were transduced with separate shRNA for GEF-H1, and RhoA activity was assessed. The knockdown efficiency was ∼79% [GEF-H1(2)]. The results in panels A to H are representative of those from 2 to 4 separate experiments. The numbers to the left of the gels are molecular masses (in kilodaltons).