Western Blotting of Cellular Extracts

Western blotting of whole-cell extracts isolated from cells in culture after FSS or extracts isolated from murine long bone was done as previously described (68 (link), 70 (link)). Equal amounts of protein were loaded and electrophoresed on 10% SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% nonfat dry milk (unless otherwise stated) and probed with the indicated primary antibodies overnight at 4°C. Antibodies were detected with the appropriate horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology) and enhanced chemiluminescence detection reagent (Bio-Rad). The antibodies used were sclerostin (R&D Systems, AF1589), α-tubulin (Sigma, T9026), detyrosinated tubulin (Abcam, ab48389), phospho-CamKII Thr²⁸⁶ (Cell Signaling Technology, 12716S), total CaMKII (Cell Signaling Technology, 11945S), and GAPDH (Millipore, MAB374). Blots were acquired using an EpiChem gel documentation system (UVP Bioimaging Systems) and analyzed using ImageJ software.