Ab100792

To obtain protein extracts, MSCs were washed with chilled PBS and lysed with RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics). The protein concentration was measured using BCA methods according to the manufacturer's instruction (P0012S, Beyotime) and 30 μg were separated by 10% SDS-PAGE gel and transferred to PVDF membranes. The protein was blocked by 5% milk for 1 h at room temperature. The membranes were incubated with specific primary antibodies against β-actin (sc-47778, Santa Cruz Biotechnology, Inc.), Wnt7a (ab100792; Abcam), OCN (ab13418; Abcam), OPN (ab214050; Abcam), RUNX2 (12556, Cell Signaling Technology, Inc.) and OSX (ab94744, Abcam) at 1:1,000 overnight at 4°C. The membranes were rinsed three times by TBST and incubated with 1:1,000 goat against rabbit (sc-2357; Santa Cruz Biotechnology, Inc.) or 1:1,000 m-IgGκBP-HRP (sc-516102; Santa Cruz Biotechnology, Inc.) horseradish peroxidase-conjugated anti- bodies for 1 h at room temperature. The bands were detected by Immobilon Western Chemiluminescent HRP Substrate (ECL, WBKLS0100; Thermo Fisher Scientific, Inc.).

Rabbit anti-mouse GPR124 ECD antibody (500 μg antibody/100 μl beads), ChromPure rabbit IgG (Jackson ImmunoResearch #011-000-003, 500 μg antibody/100 μl beads), or carrier-free anti-WNT7A antibody (abcam #ab100792, 100 μg antibody/100 μl beads) were covalently coupled to agarose beads using AminoLink Plus Immobilization Kit (ThermoFisher Scientific) according to the manufacturer’s protocol.