Comprehensive Protein Analysis Using Western Blot

Western blot analysis was conducted using the procedure previously described 49 (link). We used antibodies against, β-catenin, Lrp5, Runx2, Snail, TGFβ, MSN, PDL1, CD44 (Cell Signaling, Danvers, MA, USA), MMP9, NFATc1, cathepsin K, OPN, alkaline phosphatase, FN1 (Santa Cruz Biotechnology), CD95, RANKL, OPG (Invitrogen, Carlsbad, CA, USA), Hsp90ab1, Osteocalcin (Abcam, Cambridge, UK), KDM3A (Proteintech, Rosemont, IL, USA), and β-actin (Sigma). The levels of Hsp90ab1 and MSN in Lrp5 CM were determined using ELISA kits (MBS7700502 and MBS2124159; MyBioSource). The level of PDL-1 in EO771 cells, which were treated with 0.05, 0.1, 0.5, and 1 μg/mL of TGFβ and Lrp5 CM, was determined using a cell ELISA kit (PI62200; Invitrogen).