Fig. 2. ILK silencing induces morphological changes in BAECs. BAECs were transfected with the indicated concentration of siRNA duplexes. (A) 48 hours after the second transfection, cells were lysed in Laemmli buffer and ILK expression was analysed by western blotting using a polyclonal anti-ILK antibody (DI). Total ERK1/2 levels were monitored as a control for equal protein loading. (B) Subcellular localization and expression of ILK were determined by immunofluorescence analysis (monoclonal 65.1.9) of cells seeded on fibronectin-coated coverslips 24 hours after the second transfection. A representative photo is shown (scale bar, 20 µm). (C) Cells transfected with control or ILK siRNA were seeded on fibronectin-coated coverslips, fixed and stained with FITC-phalloidin or anti-paxillin antibody (scale bar, 20 µm). (D) Phase-contrast kymograph analysis was performed on time-lapse recordings of control or ILK siRNA-transfected cells using the kymograph function of MetaMorph. Pixel quantification was determined for 4 hours along the rectangle (length, 140 µm) depicted as a dotted line in the phase-contrast images of cells (top). (E) The surface area of spread cells was determined using MetaMorph software on at least 100 F-actin-stained cells from five different fields (100x magnification). Results (means ± s.e.m.) from a representative experiment are shown.