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. Author manuscript; available in PMC: 2010 Jan 12.
Published in final edited form as: J Biol Chem. 2007 Nov 29;283(5):2822–2834. doi: 10.1074/jbc.M704855200

FIGURE 6. MMP-9 interacts with β1-integrin after thrombin stimulation.

FIGURE 6

Panel A, FACS. U2-OS cells were stimulated with 2 units/ml of thrombin and MMP-9 expression on the cell surface measured. Panel B, immunoprecipitation. U2-OS cells were stimulated with thrombin alone or with thrombin and the PI 3-kinase inhibitor LY294002. Cells were lysed and 250µg of protein subjected to immunoprecipitation (IP) with an antibody against MMP-9 or β1-integrin, respectively. The proteins were separated from the agarose, resolved on a SDS-PAGE gel, and transferred to a nitrocellulose membrane, which was immunoblotted (IB) with an antibody against β1-integrin or MMP-9. As a control, lysates were immunoprecipitated and blotted with antibodies against MMP-9 and β1-integrin. Lower panel, control immunoprecipitation with anMMP-9isotype-specific IgG. Panel C, confocal laser scanning immunofluorescence for MMP-9 and β1-integrin. U2-OS cells were plated in 8-well culture chambers precoated with collagen I and stimulated with thrombin, fixed, and triple staining performed with MMP-9 antibody (green), β1-integrin antibody (red), and 4′,6-diamidino-2-phenylindole (blue) to visualize the nuclei. The yellow signal indicates co-localization of MMP-9 and β1-integrin. The images are representative of the most prevalent cells (bar, 20µm).