Ab264077

TSCs were lysed in radioimmunoprecipitation assay buffer (Beyotime). Immunoblotting was performed using the following rabbit primary antibodies: anti-CD9 (monoclonal; 1:2000; ab92726; Abcam, Cambridge, UK), abti-TSG101 (monoclonal; 1:2000; ab125011; Abcam), anti-Hsp70 (monoclonal; 1:1000; ab2787; Abcam), anti-tenomodulin (anti-TNMD; polyclonal; 1:1000; ab203676; Abcam), anti-collagen I (monoclonal; 1:1000; ab270993; Abcam), anti-scleraxis (anti-SCXA; polyclonal; 1:500; DF13293; Affinity Biologicals, Ancaster, ON, Canada), anti-alkaline phosphatase (anti-ALP; polyclonal; 1:1000; DF6225; Affinity Biologicals), anti-runt-related transcription factor 2 (anti-Runx2; monoclonal; 1:1000; ab264077; Abcam), anti-SMAD2/3 (monoclonal; 1:1000; 5678S; Cell Signaling Technology, Danvers, MA, USA), anti-phospho (p)-SMAD2/3 (monoclonal; 1:1000; 8828S; Cell Signaling Technology), anti-SMAD1/5/9 (polyclonal; 1:500; AF0614; Affinity Biologicals), anti-phospho-SMAD1/5/9 (polyclonal; 1:1000; AF8313; Affinity Biologicals), and anti-β-actin (monoclonal; 1:5000; ab8226; Abcam). Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; BA1055; Boster, Wuhan, China) was used as the secondary antibody. A chemiluminescence imaging system (ChemiScope 6200T, Clinx Science Instruments, Shanghai, China) was used for detection.

MC3T3-E1 cells were induced in osteogenic induction medium for 3 days. Cells were lysed by radioimmunoassay (RIPA; Beyotime), centrifuged and the supernatant was collected, and the total protein concentration was quantified by BCA kit (Beyotime). Total protein (20 μg) was separated by SDS-PAGE (New Cell and Molecular Biotechnology Limited, Suzhou, China) and transferred to a nitrocellulose membrane. The cells were incubated with the appropriate primary antibody for 16 h at 4°C after blocking, and the antibody of Runx2 (1 : 1000), Osterix (1 : 1000), total GSK-3β (1 : 1000), pser 9-gsk-3 β (1 : 1000), and β-catenin (1 : 5000) was purchased from Abcam (ab264077, ab209484, ab93926, ab107166, and ab32572). Membranes were washed with TBST (BP-G0004, CWBiotech, Beijing, China), then incubated with secondary antibody and observed with chemiluminescent HRP substrate (WBKLS0500, Millipore Corp.).