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Figure 1: CD44 knockout reduces the levels of active MMP9 but increases MT1-MMP in osteoclasts. (a) Zymogram analysis. Conditioned media from CD44−/− (lane 2) and WT osteoclasts (lane 3) were analyzed by zymogram analysis. Recombinant MMP9 protein (UBI) was used as an identification marker. (b) Reverse zymography. The lower half of the zymogram was cut and incubated with culture media to digest the gelatin in the gel. Subsequently, the gel was stained with Coomassie blue to detect TIMP2 protein as described in the Methods-section. Dark TIMP2 band (indicated by arrows) was observed in the light background. (c–e) Immunoblotting analyses. Soluble or secreted levels of MT1-MMP and TIMP 2 in the conditioned media of CD44−/− (lane 1) and WT (lane 2) osteoclasts were determined by immunoblotting analysis with relevant antibody in succession after stripping the blot. Finally the blot was stained with a Coomassie blue stain to determine the amount of protein loaded in each lane (e). (f) and (g) FACs analysis (f) and MT1-MMP activity assay (g). (f) A representative histogram is shown for CD44−/− and WT osteoclasts. Each FACs analysis was performed in quadruplicates. Mean fluorescence intensity of MT1-MMP from two different osteoclast preparations is shown in Figure 1(f). (g) Concentrated conditioned media and MT1-MMP immunoprecipitates pulled down with streptavidin agarose were subjected to the MT1-MMP activity assay to determine the soluble and cell surface (membrane) associated enzyme activity, respectively. ** versus WT osteoclasts in (f) and (g). Results shown in (g) are representative of three different experiments with three different osteoclast preparations.