Figure 5: MT1-MMP regulates the activity of MMP9. (a–d) The effects of various treatments on MMP9 activity. Equal amounts of membrane proteins (50 μg) from osteoclasts untransfected (−) (lane 2) or transfected with SiRNA (Si, lane 3) and ScRNAi (Sc, lane 4) to MT1-MMP were used for gelatin zymography analysis (a) and MMP9 activity assay in vitro (d). Lysates made from osteoclasts treated with TIMP2 (T2) were also used for MMP9 activity assay in vitro (d). To ensure that equal protein amount was used for zymogram analysis (a), duplicate polyacrylamide gel was run with same amount of protein and stained with Coomassie blue staining (b). Cell extracts from osteoclasts treated with ScRNAi (Sc) and SiRNA (Si) to MT1-MMP were subjected to immunoblotting analysis with an MMP9 antibody to detect total cellular levels of MMP9 ((c); top panel). Stripping and reprobing of the same blot with an antibody to GAPDH was used as loading control (bottom panel in (c)). (e) The effects of various treatments on MT1-MMP activity. MT1-MMP activity was determined in the membrane fraction of osteoclasts subjected to various treatments as indicated as follows: ScRNAi (Sc) and SiRNA (Si) to MT1-MMP and MMP9, TIMP1 (T1), and TIMP2 (T2). Untransfected and CD44-null osteoclasts are indicated as (−) and CD44−/−, respectively. Values in (d) and (e) are mean ± SE from three different experiments. ** and *** compared to controls ((−) and Sc). The results shown are representative of three different experiments.