Figure 4.

Fibronectin matrix removal blocks Rho-mediated tyrosine phosphorylation of focal contact proteins FAK and paxillin. (A) HUVECs were treated for 4 h with 20 μM III1-C in serum-free medium to remove fibronectin matrix (+) or with the medium alone (−). Where indicated, LPA cells were stimulated with 5 μM LPA for 15 min before lysis. As a control for the effects of III1-C without removing fibronectin matrix, 20 μM III1-C was added for 15 min in absence (III1-C) or presence of 5 μM LPA (III1-C + LPA). FAK or paxillin were immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody (PY20). The blots were reprobed with anti-FAK or anti-paxillin antibodies to show that similar protein levels were immunoprecipitated and to detect FAK that had coimmunoprecipitated with paxillin. (B) Suspended HUVECs were treated for 1 h with (+) or without (−) 20 μM III1-C in serum-free medium before plating on dishes coated with 10 μg/ml fibronectin, vitronectin, or laminin. The cells were lysed after 30 min, and FAK or paxillin was immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody (PY20). The experiments were carried out three to five times, and a representative result is shown.