A2692

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).