FIG. 4.

Integrin signaling and stress fibers act upstream of Ras to sustain ERK activity. (A) Quiescent hα5-3T3 cells were pretreated with DMSO or ML-7, plated on dishes coated with either fibronectin or poly-l-lysine (PLL), and stimulated with 10 ng of bFGF/ml. Cells were collected at the indicated times, lysed, and analyzed for total and GTP-loaded Ras. Identically treated cells were analyzed for Raf-1 activity and the level of total Raf-1 (see Materials and Methods). Total cell lysates were also used to monitor the activities of MEK and ERK by immunoblotting for phosphorylation of MEK at S222 and dual phosphorylation of pERK, respectively. (B) hα5-3T3 cells were transiently transfected with empty vector or FRNK, serum starved for 24 h (0), and then held in suspension for 1 h (lanes S), prior to stimulation on fibronectin (FN)-coated dishes with 10 ng of bFGF/ml. Collected cells were lysed and analyzed by immunoblotting with antibodies to pY397 FAK, FAK, pERK, ERK, cyclin D1, and cdk4 (loading control).