The effect of integrin αv inhibitor on RANKL expression and osteoclast differentiation. A-C: Western blot analysis. Equal amount of total cellular lysates (50 μg protein; A, lanes 1 and 2) and conditioned media (CM-20 μg protein; lanes 3 and 4) were immunoblotted with a RANKL antibody. CM was used to detect the secreted levels of RANKL. The blot in A was reprobed with a GAPDH antibody after stripping (B). GAPDH level was used as a control for loading. The loading control for the CM is shown by the use of Coomassie blue staining of a gel ran in parallel (C). D and E. The effect of CM on osteoclast differentiation in vitro is shown. TRAP-positive osteoclasts are stained in dark purple. Images were captured (X 200) with an inverted phase contrast microscope. The results shown are representative of three independent experiments.