Analysis of RANKL expression level in PC3 cells knockdown of CD44. A and C. Equal amount of total cellular lysates (50 μg protein; A) and conditioned media (CM-20 μg protein; C) were immunoblotted with a RANKL antibody to detect RANKL protein. CM was used to detect the secreted RANKL protein. B and D. The blot in A was stripped and reprobed with a GAPDH antibody. Equal level of GAPDH protein was observed (B). The loading control for the CM was shown by the use of Coomassie blue staining of a gel ran in parallel (D). E-G. 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.