Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody

Western blotting was performed as previously described.
²³ (link) RIPA protein extraction reagent (Beyotime, Beijing, China) was used to lyse BMSCs, followed by treatment with protease (Beyotime, Beijing, China) and phosphatase inhibitors (Beyotime, Beijing, China). The concentrations of isolated proteins were determined using a BCA Protein Assay Kit (Beyotime, Beijing, China). Thirty micrograms of total protein was electrophoretically separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE, Solarbio, Beijing, China) and then transferred to polyvinylidene difluoride membranes (PVDF, Solarbio, Beijing, China). The membranes were incubated with 5% bovine serum albumin (BSA, Solarbio, Beijing, China) for 2 h. Primary antibodies against β‐catenin, GSK‐3β, DKK1, RUNX2, OSX, OPN, OCN and TCF‐1 were applied to the membranes and incubated overnight at 4°C. Details of the manufacturers, dilutions and product numbers of the antibodies are shown in Table S1. The membranes were then incubated with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit secondary antibody (Abbkine, Wuhan, China) for 2 h at 37°C. Subsequently, the bands were visualized using an Enhanced Chemiluminescence Kit (Epizyme Biomedical, Shanghai, China), and the results were evaluated using Quantity One Imaging Software (Bio‐Rad, CA, USA).