Cocktail of protease phosphatase inhibitors

TMSCs were washed twice with PBS and lysed in RIPA lysis buffer supplemented with cocktail of protease/phosphatase inhibitors (Thermo Fisher Scientific). Supernatants were collected after centrifugation at 12,000 rpm for 10 min. Humerus was frozen rapidly using liquid nitrogen immediately after dissection. Frozen humerus was homogenized using a mortar and pestle on dry ice. Whole protein of bone was extracted in ice by RIPA lysis buffer supplemented with cocktail of protease/phosphatase inhibitors for 20 min. Supernatants were collected after centrifugation at 12,000 rpm for 10 min and protein concentration was measured using BCA protein assay kit (Pierce). Primary antibodies used were anti-Wnt16 (Thermo Fisher Scientific), anti-RUNX2, anti-osteocalcin, anti-CathepsinK (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and anti-beta actin (Sigma-Aldrich) antibodies. Secondary antibodies used were HRP conjugated antibodies. Protein bands were visualized with ECL™ Prime Western Blotting Detection Reagent (Amersham) in GBOX chemi XX9 (Syngene).

12-O-Tetradecanoylphorbol 13-acetate (PMA), human recombinant TNF-α, dimethyl sulfoxide (DMSO), 2-mercaptoethanol, sodium chloride, phenylmethylsulfonyl fluoride (PMSF), TAPI-1, DAPT, and β-secretase inhibitor IV (C3) were purchased from Sigma-Aldrich (St. Louis, MO, United States). APOE2 and APOE4 were obtained from Biovision (Milpitas, CA, United States). Go 6983, Ro 32-0432, Wortmannin and LY294002 were purchased from Tocris (Minneapolis, MN, United States). All antibiotics, fungizone, PBS, culture media, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, United States). The MPER reagent and the cocktail of protease/phosphatase inhibitors were purchased from Thermo Fisher Scientific (Waltham, MA, United States).