Cells were lysed with radioimmunoprecipitation assay buffer and proteinase inhibitors, and amount of total protein were measured with Bio-Rad protein assay (Bio-Rad, CA, USA) according to the manufacturer’s protocol. The samples were prepared with 4× Laemmli sample buffer and 2-mercaptoethanol (Bid-Rad), and SDS–polyacrylamide gel electrophoresis was performed with TGX precast gels (Bio-Rad) and the Mini-PROTEAN electrophoresis system (Bio-Rad). The gel was then transferred to a mini polyvinylidene difluoride membrane with the Trans-Blot Turbo transfer system (Bio-Rad). The membrane was blocked with 5% skim milk in 1× Tris buffered saline with Tween 20 (TBS-T) buffer, and primary and horseradish peroxidase (HRP)–conjugated secondary antibodies were diluted in CanGetSignal solution (TOYOBO, Japan). Following incubation with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific), chemiluminescence signals were detected with LAS4000 imager (GE Healthcare). Antibodies were obtained as follows: Runx2 (8486S, 1:1000, Cell Signaling Technology Inc., MA, USA), Sp7 (ab22552, 1:1000, Abcam), and HRP-conjugated anti-rabbit immunoglobulin G (IgG) (NA934, 1:10,000; GE Healthcare, IL, USA).

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