Investigating Effects of SPARC on Cell Adhesion

Cancer cells were seeded onto cell culture dishes coated with collagen I. In addition, either the collagen substrate or the culture medium was supplemented with 750 ng/mL rSPARC or equal amounts of PBS + 0.1% BSA as control. After 24 h incubation, cells were lysed, and proteins extracted for western blotting as described in [43 (link)]. Briefly, protein was extracted using HEPES lysis buffer supplemented with protease inhibitors cocktail (Complete, Roche, Germany) and phosphatase inhibitors. 10 µg protein per lane were analyzed by western blotting. The antibodies used recognized integrin β1 (1:1000; 4706, Cell Signaling Technology, Danvers, MA, USA), FAK (1:1000; 71,433, Cell Signaling Technology), p-FAKTyr397 (1:1000; 8556, Cell Signaling Technology), E-cadherin (1:1000; 3195, Cell Signaling Technology), and β-actin (1:5000, AC-15, Sigma, St. Louis, MO). Detection was achieved by chemiluminescence and band intensity was determined using ImageJ software. Uncropped scans of all blots can be found in Figure S5.