Sca 1

Western blot analysis was performed using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis system. Protein samples (20 µg) were resuspended in reduced sample buffer, electrophoresed on a 7.5-10% Tris gel with Tris running buffer, blotted onto PVDF membranes, and sequentially probed with primary antibodies against RUNX2 (Cat. no. 12556; 1:1,000; Cell Signaling Technology), OCN (Cat. no. sc-365797; 1:1,000; Santa Cruz Biotechnology, Inc.), eukaryotic translation initiation factor 4E-binding protein 1 (4E/BP1; Thr37/46; Cat. no. 9644; 1:1,000; Cell Signaling Technology), phospho-S6 ribosomal protein (P-S6; S235/S236; Cat. no. sc-293143; 1:1,000), PPARγ (Cat. no. sc-7273; 1:1,000) (both from Santa Cruz Biotechnology, Inc.), C/EBPβ (Cat. no. 3802; 1:2,000), Sca-1 (Cat. no. 9664; 1:4,000), CD29 (Cat. no. 4706; 1:2,000), CD45 (Cat. no. 13917; 1:6,000), CD11b (Cat. no. 14271; 1:3,000) and β-actin (Cat. no. 3700; 1:2,500) (all from Cell Signaling Technology). Horseradish peroxidase-conjugated goat anti-rabbit (Cat. no. SAB4600223; 1:1,000) or anti-mouse (Cat. no. SAB4600004; 1:1,000) antibodies (both from Sigma) were added, and secondary antibodies were detected using enhanced chemiluminescence (ECL Plus; General Electric Healthcare, Milwaukee, WI, USA).