Protein Expression Analysis in Osteogenic Differentiation

Total cellular protein (Signalway Antibody LLC, USA) and nuclear protein (Shanghai Sangon Biotechnology Co., Ltd, China) were extracted using the corresponding kit. The sample concentration was determined by a BCA protein assay kit (Beyotime, China). After being mixed with loading buffer, the protein sample was boiled at 95 °C for 5 min. The protein sample was separated by SDS polyacrylamide gel electrophoresis and transferred to PVDF membrane. Later, the blots were blocked in 5% fat-free skimmed milk for one hour at room temperature, followed by incubation with primary antibodies (Huabio, China) against Osterix (1:1000, ER1914-47), COL-I (1:1000, ET1609-68), OCN (1:1000, ER1919-20), Runx2 (1:1000, ET1612-47), ALP (1:1000, ET1601-21), ERK1/2(1:5000, ET1601-29), pERK1/2 (1:1000, ET1603-22), STAT3(1:1000, ET1607-38), pSTAT3(1:1000, ET1603-40), and GAPDH (1:1000, ET1601-4 ) overnight at 4 °C. After a three-time wash in TBST solution, membranes were probed with goat anti-rabbit IgG conjugated secondary antibody (Hubio, China) for one hour at room temperature. The blots were cut prior to hybridization with antibodies during blotting. The membranes were processed with enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and visualized.