302392.fig.003
Figure 3: MT1-MMP knockdown reduces CD44 surface expression. (a) and (b) Immunoblotting analyses with an antibody to MT1-MMP (a) and MMP9 (b) were performed in total cellular lysates (50 μg protein) made from osteoclasts treated with a scrambled RNAi (Sc; lane 1 in (a) and (b)) or SiRNA sequences to MT1-MMP (lanes 2 and 3 in (a)) and MMP9 (lane 2 in (b)). The blots in (a) and (b) was reprobed with an antibody to GAPDH after stripping (bottom panel in (a) and (b)). GAPDH level was used as a control for loading. ((c) and (d)) Immunoblotting analysis of surface expression of CD44s in osteoclasts treated with a SiRNA (Si) or scrambled RNAi (Sc) to MT1-MMP (c) and MMP9 (d). Equal amounts of proteins (50 μg protein) prepared from osteoclasts treated as indicated in (c) and (d) and surface labeled with NHS-biotin were immunoprecipitated with an antibody to CD44 (lanes 1–3 in (c); lanes 1 and 2 in (d)). Lysates from scrambled RNAi-treated osteoclasts were immunoprecipitated with a nonimmune serum (NI; lane 4 in (c); lane 3 in (d)). Immunoprecipitates were probed with streptavidin-HRP to determine the surface levels of CD44 protein. Blot in (c) and (d) was stripped and reprobed with an antibody to CD44 to determine the total cellular levels of CD44 protein ((e) and (f)). (g) To ensure that equal protein amount was used for immunoprecipitation, duplicate polyacrylamide gels were run with 50 μg total protein and stained with Coomassie blue staining.