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Figure 2: CD44 knockdown reduces membrane localization of MT1-MMP (~63 kDa) in osteoclasts. (a) Membrane fraction (M; lanes 1 and 3) and conditioned medium (CM; lanes 2 and 4) of osteoclasts isolated from WT (lanes 1 and 2) and CD44−/− (lanes 3 and 4) mice were analyzed by immunoblotting with an MT1-MMP antibody (top panel). Well-characterized protein standards (indicated on the right side of the figure) were used to estimate the approximate molecular weight of MT1-MMP in the membrane fraction and conditioned media. Estimated approximate molecular masses of MT1-MMP (~65, 55, and 45) are indicated on the left side of the figure. Membranes were stripped successively and blotted with an antibody to actin (middle panels) and stained with Coomassie blue (bottom panels) as loading controls for membrane fraction and conditioned medium, respectively. Data shown are representative of three independent experiments with similar results. (b) and (c) TIMP2 binds active MT1-MMP form (~55 kDa) in the membrane and conditioned medium of CD44−/− osteoclasts. Equal amount of protein from membrane fraction (50 μg; M) and conditioned medium (25 μg; CM) from WT (lanes 1 and 3) and CD44−/− (lanes 2 and 4) was used for immunoprecipitation with an antibody to MT1-MMP and immunoblotted (IB) with an antibody to TIMP2 (top panels in (b)). Membranes were stripped and blotted with an antibody to MT1-MMP ((c); bottom panels). Protein A-HRP was used as secondary antibody as described previously [31]. This analysis provided specific signals corresponding to 55 kDa. Data shown are representative of three independent experiments with similar results.