Fig. 4.

Ras was not essential for the disassembly of adherens junctions inducedby integrin engagement. (A) BAEC monolayers were incubated for 5 min with control medium (control), Beads + Pl, Beads + Fn, or Beads + Fn + PP1. Ras activity was assayed as described in Experimental Procedures. (A Upper) Active Ras shows the Ras activities. (A Lower) Total Ras shows the Ras protein levels. (B and C) BAEC monolayers were infected by control vector Ad5, Ad_RasV12, or Ad_RasN17. After a 4-h incubation with serum-free medium, the cells were stained with anti-γ-catenin antibody (B) or subjected to IP with anti-VE-cadherin antibody and IB with appropriate antibodies to detect the association of VE-cadherin and adherens junction proteins (C). (D) BAEC monolayers were infected by Ad_RasN17 before being subjected to Beads + Pl or Beads + Fn for 20 min. γ-catenin is shown in red and beads in blue. (E) BAEC monolayers were infected by Ad5 or Ad_RasN17 before being subjected to the transfection of BSA or active Src proteins. The images show the γ-catenin staining. (F) BAEC monolayers treated with PP1 (10 μM), Ad_RasN17, or kept as control were treated with Beads + Pl or Beads + Fn. Cell lysates from various samples were probed for phospho-ERK (Upper) and ERK2 (to show comparable protein loading among various samples, Lower). The results are representative of three separate experiments.