Proteasome and phosphatase inhibitors

Cells were lyzed in RIPA buffer supplemented with proteasome and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with antibodies specific to GAPDH (1:1,500; CST#5174, Cell Signaling Technology, Danvers, MA, United States), COL1A1 (1:1,000; ab34710, Abcam, Cambridge, United Kingdom), RUNX2 (1:1,600; ab192256, Abcam), active β-catenin (1:1,000; ab246504, Abcam) or total β-catenin (1:1,000; ab223075, Abcam). A stripping method was used to measure the two antibodies of same molecular weight. After washing four times (5 min each time) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Beyotime) for 1 h at room temperature. After washing three times (5 min each time) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United).