Fig. 5.

Anastellin causes inactivation of β1 integrin. Microvessel cells were treated with vehicle (control), 20 µM anastellin or the control fibronectin module FnIII₁₃ for 60 minutes. (A) Cells were washed, fixed and stained for active β1 integrin using the monoclonal antibody 9EG7. Nuclei were counterstained with Hoechst 33258. (B) NIH ImageJ software was used to quantify the number of β1-containing focal adhesions obtained from at least five fields of view. Data are shown normalized to control values. Data averaged over five independent experiments are represented as the mean±s.e.m.; *P<0.001. (C) Cells were stained for both actin (red) and active β1 integrin (green) using phalloidin and the 9EG7 antibody, respectively. Control cells show β1 integrin localized at actin termini in focal adhesions (arrowheads). (D,E) Microvessel cells were stained with a monoclonal antibody directed against integrin β5 and quantified as in B. (F) Microvessel cells were dual stained for β1 (9EG7, green) and α5 (AB1949, red) integrin subunits. Merged images are shown on the right. Scale bars: 25 µm.