Sclerostin

Western blot analysis was conducted using a previously described protocol.^{39 (link)} We used antibodies against ANXA1, β-catenin, caspase 3, Lrp5, Lrp6, Runx2, Sclerostin, Snail, TGFβ, NFATc1, cathepsin K (all from Cell Signaling, Danvers, MA, USA), DMP1, ANXA6, CXCL5 (all from Abcam, Cambridge, MA, USA), M-CSF, MMP9, OPN, TPM4 (all from Santa Cruz, Dallas, TX, USA), WISP1 (R&D systems, Minneapolis, MN, USA), β-actin (Sigma, Saint Louis, MO, USA), LIMA1, Trail (both from Novus, Centennial, CO, USA), p53, CXCL1 (both from Invitrogen, Carlsbad, California, USA), and DSP (ProteinTech, Rosemont, IL, USA). The expression levels of Sclerostin and Lrp5 in CM were detected by ELISA (My BioSource, San Diego, CA, USA). Proteins isolated from A5 osteocyte CM, Y4 osteocyte CM, and osteoclast control CM (RAW264.7 cells) were analyzed with an HF Hybrid Quadrupole Orbitrap mass spectrometer. Among the 549 identified proteins, 49 proteins had higher expression levels in A5 CM than in Y4 CM and control CM. Among these proteins, 11 (p53; SPARC = osteonectin; TPM1, TPM4 = tropomyosin 1 and 4; ANXA1, ANXA6 = annexin A1 and A6; FMOD = fibromodulin; OGN = osteoglycin; DSP = desmoplakin; AHNAK = desmoyokin; and LIMA1 = LIM domain actin-binding protein 1) were identified as potential tumor suppressors.