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. 2009 Sep 17;284(46):31764–31775. doi: 10.1074/jbc.M109.038547

FIGURE 3.

FIGURE 3.

Cell adhesion activity of rLG1 is mediated by heparin binding activity. A, cell adhesion to laminin, rLG proteins, and Ln2-P3 in PC12 cells pretreated with EDTA or integrin β1 antibody. PC12 cells were pretreated with 5 mm EDTA or 10 μg/ml of monoclonal function-blocking antibody against the integrin β1 subunit for 15 min at 37 °C and then seeded on plates precoated with laminin (LN; 5 μg/ml), rLG proteins (25 μg/ml), or Ln2-P3 (22 μg/cm2) for 1 h in serum-free medium. B, cell adhesion to laminin, rLG proteins, and Ln2-P3 in PC12 cells pretreated with heparin for 10 min at room temperature. The assay conditions were the same as described in A. C and D, adhesion of heparin to rLG1 and Ln2-P3. Enzyme-linked immunosorbent assay plates were coated with rLG proteins or synthetic peptides for 12 h at 4 °C, blocked with 3% BSA for 2 h at 37 °C, and then incubated with 10 ng/well of biotinylated heparin for 1 h at 37 °C. The biotinylated heparin bound to rLG proteins or synthetic peptides was detected using streptavidin-conjugated horseradish peroxidase. E, inhibition of cell adhesion to rLG1 and Ln2-P3 in cells pretreated with heparan sulfate and dermatan sulfate. PC12 cells were pretreated with 100 μg/ml of heparin, heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate A (CSA), dermatan sulfate (DS), chondroitin sulfate C (CSC), or de-N-sulfated heparin (de-N-s hep) for 10 min at room temperature and seeded on plates precoated with rLG1(25 μg/ml) or Ln2-P3 (22 μg/cm2) for 1 h in serum-free medium. The number of adherent cells was quantified by cell counting. F, inhibition of cell adhesion to rLG1 and Ln2-P3 in cells pretreated with heparitinase I or chondroitinase ABC for 90 min at 37 °C. The assay conditions were the same as described in E. Values are expressed as a percentage of the value for cells pretreated without EDTA or integrin β1 antibody, heparin, glycosaminoglycans, or enzymes (None) in A, B, E, and F (mean ± S.D., n = 3). Values are expressed as the mean ± S.D. (n = 4) in C and D. *, p < 0.01; **, p < 0.05.