A5786

Total protein of MC3T3 cells were harvested with RIPA lysis buffer and quantified with a BCA assay kit (Beyotime). Proteins were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% bull serum albumin in Tris buffered saline with Tween 20 and incubated with the following primary antibodies at 4 °C overnight: cyclin D1 (1:500, A2708, ABclonal, Wuhan, China), SP7 (1:1000, ab94744, Abcam, Shanghai, China), OCN (1:650, A5786, ABclonal), Runx2 (1:500, A2851, ABclonal), P27^Kip1 (1:500, A2692, ABclonal), and β-actin (1:100000, AC026, ABclonal). Horseradish peroxidase-conjugated anti-rabbit IgG (H + L) secondary antibodies were added (1:3000, AS014, ABclonal) and incubated at 25 °C for 1 h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech, Shanghai, China) by Tanon 5200 (Tanon, Shanghai, China).

Total protein of MC3T3 cells were harvested with RIPA lysis buffer and quantified with a BCA assay kit (Beyotime). Proteins were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% bull serum albumin in Tris buffered saline with Tween 20 and incubated with the following primary antibodies at 4 °C overnight: cyclin D1 (1:500, A2708, ABclonal, Wuhan, China), SP7 (1:1000, ab94744, Abcam, Shanghai, China), OCN (1:650, A5786, ABclonal), Runx2 (1:500, A2851, ABclonal), P27^Kip1 (1:500, A2692, ABclonal), and β-actin (1:100000, AC026, ABclonal). Horseradish peroxidase-conjugated anti-rabbit IgG (H + L) secondary antibodies were added (1:3000, AS014, ABclonal) and incubated at 25 °C for 1 h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech, Shanghai, China) by Tanon 5200 (Tanon, Shanghai, China).

The tissue sections were fixed with acetone for 5 min and air-dried for 10 min. The sections were then immersed in 0.3% H₂O₂/PBS for 5 min to remove peroxidase activity. After treating with blocking solution for 1 h, primary antibodies were applied and incubated for 1 h, followed by secondary antibodies (HRP-conjugated goat anti-rabbit/mouse IgG), reacting for another hour. The sections were then stained with DAB for 5 min and rinsed with distilled water for another 10 min. The primary antibodies used in this experiment were TGF-β (sc-146, Santa Cruz), α-SMA (A2547, Sigma-Aldrich), Collagen I (A5786, ABclonal), IL-6 (sc-1265, Santa Cruz), IL-17A (A12454, ABclonal), and TNF-α (A11534, ABclonal).