Cw2333s

Cultured cells were washed thoroughly with cold PBS and lysed in RIPA buffer (Cwbio, CW2333S) containing 1% PMSF (Beyotime, ST505) and a phosphatase inhibitor (Beyotime, ST637). The lysate was centrifuged at 4 °C for 30 min, and the protein concentration in the supernatant was measured with a BCA assay kit (Cwbio, CW0014S). Equal amounts of each sample were diluted in 5 × sodium dodecyl sulfate loading buffer (Beyotime, P0015), separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Beyotime, P0012) and then transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010). Membranes were blocked with 5% nonfat milk in TBST (150 mM NaCl, 0.05% Tween-20, and 50 mM Tris–HCl, pH 7.5) for 1 h at room temperature and incubated overnight at 4 °C with antibodies against GAPDH (1:1000, Cell Signaling Technology, 5174), IL-8 (1:1000, Abcam, ab235584), c-Jun (1:1000, Cell Signaling Technology, 9165), Runx2 (1:1000, Cell Signaling Technology, 8486), Osterix (1:1000, Abcam, ab209484) and OCN (1:1000, Abcam, ab133612). The membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (BOSTER, BA1054) or anti-mouse IgG (BOSTER, BA1050) diluted 1:3000 at room temperature for 1 h. Immobilon Western chemiluminescent HRP substrate (Millipore, WBKLS0050) was used to visualize the membranes. ImageJ was used to quantify band intensities.