A flow cytometory analysis using phycoerythrin conju gated mouse anti CD14 mAb showed that purity of the CD14 monocytes thing was more than 98% in each experiment. The purified CD14 monocytes were cultured in 96 wells in alpha minimum essential medium with 10% FBS and incubated with M CSF and soluble RANKL with or without bioactive recombinant CTGF. The medium was replaced with fresh medium three days later and the cells were stained for tartrate resistant acid phosphatase expression using a commercial kit after incubation for seven days. The number of TRAP positive multinucleated cells in three randomly selected fields examined at 100�� magnification of the Inhibitors,Modulators,Libraries total number of TRAP positive MNC per well were counted as oste oclasts under light microscopy.

For immunoblotting and immu noprecipitation analysis, osteoclasts were initially differentiated by M CSF and sRANKL without CTGF for seven days. Then, recombinant CTGF was added into the cultures and incubated at 5, 15, 60, and 120 minutes in the presence or absence of anti CTGF antibody. The cells were washed and col lected for making cell extracts for subsequent Inhibitors,Modulators,Libraries immunoblotting and immunoprecipitation assays. Immunoblotting and immunoprecipitation MH7A and OUMS 27 cells were centrifuged at 200 g for 30 min. Cell pellets were then resuspended directly in lysis buffer containing 150 mM NaCl, 1 mM MgCl26H2O, 80 mM Tris HCl, 0. 1% NP 40 and Complete Protease Inhibitor cocktail. The protein concentrations in the lysates were determined using the Protein DC Assay Kit to ensure equal loading of proteins in each SDS PAGE lane.

After determining the protein concentration, lysates were mixed with an equal volume Inhibitors,Modulators,Libraries of 2 gel sample buffer contain ing 6% sodium dodecyl sulfate, 20% glycerol, 10% mercap toethanol, 0. 02% bromophenol blue and Complete Protease Inhibitor cocktail. Lysates were Inhibitors,Modulators,Libraries stored at 80 C until use. The equivalent of 10 g total lysate protein was loaded onto each lane of 12. 5% SDS PAGE gels, separated by electrophoresis and then transferred to nitrocellulose membranes using a Semi Dry Trans Blot apparatus. After blocking, immunoblotting was performed using polyclonal goat anti human CTGF antibody at 1 2,000 and monoclonal mouse anti human actin antibody at 1 500 dilution. The detec tion of bound antibodies was achieved using horseradish per oxidase conjugated anti goat IgG antibody and anti Inhibitors,Modulators,Libraries mouse IgG antibody used at 1 5,000 and 1 2,500 dilution respectively, in combination with enhanced chemiluminescence.

Cell extracts of the osteoclasts stim ulated with or without recombinant CTGF were prepared with the same condition for subsequent immunoblotting and immu noprecipitation assays. For immunoprecipitation, 5 g of mouse anti human integrin V 3 antibody were incubated at 4 sellectchem C overnight with protein G conjugated agarose beads.