FIG. 9.

Effects of inhibition of Dok-1 function on the activity of Rho. (A) B16F10 cells transfected with the empty vector (Cont, [control]) or expressing DokPH+PTB (PH+PTB5) were detached from their culture dishes and then either maintained in suspension (Susp) or replated on fibronectin-coated dishes (Adh). After incubation of cells for 30 min in MEM supplemented with 1% FBS, the active form of Rho was precipitated from cell lysates with a GST fusion protein containing the Rho-binding domain of Rhotekin. The resulting precipitates were then subjected to immunoblot analysis with a MAb to RhoA (top). Whole-cell lysates were also directly subjected to immunoblot analysis with the same MAb to determine the total amount Rho (bottom). (B) The same two cell lines were deprived of serum for 12 h and then incubated for 1 min in the absence (−) or presence (+) of 4 μM LPA (Sigma). The active form of Rho was then precipitated and analyzed as in panel A.