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Fig. 2. Interaction of 
9β1 integrin with neurotrophins in an adhesion assay. (A) Adhesion of 9SW480- (circles) and mock-SW480- (triangles) transfected cells to immobilized mNGF (filled symbols) and pro-NGF (open symbols). mNGF or pro-NGF was immobilized in a 96-well microplate in PBS by overnight incubation at 4°C. Cells were labeled with CMFDA and added to the wells, previously blocked by BSA. Incubation was performed at 37°C for 30 minutes in HBSS buffer containing calcium and magnesium. After washing off unbound cells, Triton X-100 was added to the wells and the plate was read using a fluorescence microplate reader. The number of adhered cells was calculated from the standard curve prepared in parallel in the same plate from a known number of cells. (B) Adhesion of 9SW480 cells (filled bars) and mock-SW480 cells (open bars) to immobilized neurotrophins, growth factors, monoclonal antibody and adhesion molecule. All proteins were immobilized on 96-well plates at a concentration of 10 µg/ml in PBS by overnight incubation at 4°C. Adhesion experiments were performed as described above. (C) Effect of various monoclonal antibodies and snake venom disintegrins on adhesion of 9SW480 cells to immobilized (10 µg/ml) mNGF. Monoclonal antibodies Y9A2 (anti-9β1), Lia1/2 (anti-β1), SAM-1 (anti-5) LM609 (anti-vβ3), or snake venom disintegrins at concentrations of 10 µg/ml, were preincubated with CMFDA-labeled cells for 15 minutes before adding to the wells for adhesion. Adhesion experiments were performed as described above. (D) Effect of mNGF and VLO5 on expression of LIBS epitope on the β1 subunit of integrin in 9SW480 cells. Anti-LIBS, anti-human monoclonal antibody (B44), was immobilized in 96-well plates at a concentration of 10 µg/ml in PBS, overnight at 4°C. VLO5 or mNGF at a concentration of 1 µM was preincubated with CMFDA-labeled 9SW480 cells for 15 minutes before addition to the wells for adhesion. Adhesion experiments were performed as described above. Error bars indicate the standard deviation from three independent experiments.
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