Ab66721

Cell lysates were prepared by extracting proteins using a lysis buffer ((20 mM Tris-HCl, pH 7.5 150 mM NaCl, 1 mM ethylenediaminetetracetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton, and 1 mM phenylmethylsulfony fluoride (PMSF)), supplemented with protease inhibitors. The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare Amersham^TM, Piscataway, NJ, USA). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline and then incubated with the primary antibodies (Wnt 10b: ab 66721, abcam, Burlingame, CA, USA; Wnt 16: ab 64461, abcam, Burlingame, CA, USA) for 1 h at room temperature. The blots were then developed with a peroxidase-conjugated secondary antibody, and the proteins visualized using enhanced chemiluminescence procedures (Amersham, Arlington Heights, IL, USA) according to the manufacturer’s instructions.

The osteoclasts were cultured on 22 × 22-mm² glass coverslips for 4 days as previously described [63 (link)]. The osteoclasts were then treated with 400 nM cinacalcet for 18 h. They were washed with phosphate buffered saline (PBS) and further fixed in 4% paraformaldehyde for 10 min. Then, permeabilization was completed with PBS containing 0.05% Triton X-100 for 1 h. The treated cells were incubated with an antibody specific for Wnt10b (abcam ab66721, USA) in 1% bovine serum albumin in PBS overnight at 4 °C. Cell Navigator™ F-Actin Labeling Kit (Cat No:22663, AAT Bioquest, Inc., Sunnyvale, CA, USA) was used (15 min) to visualize the F-actin distribution. The nuclei were counterstained with Nuclear Red™ DCS1 (1:1000 dilution; AAT Bioquest, Sunnyvale, CA, USA). A confocal microscope equipped with a differential interference contrast optical path (LSM 510, Zeiss, Göttingen, Germany) was used for further imaging. The osteoclasts were considered as successfully cultured if the multi-nucleated cells had more than three nuclei and when more than half of the actin ring was labelled [64 (link)].