Horseradish peroxidase conjugated anti mouse or anti rabbit secondary antibodies

After 7 days of osteogenic induction, the transfected SCAP were harvested and lysed with RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Shanghai, China) on ice. Total protein concentrations were measured by the BCA Protein Assay Kit (Solarbio). The proteins in all samples were separated by SDS-PAGE gels, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States). After blocking in 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. Primary antibodies included DSPP (Santa Cruz Biotechnology, Santa Cruz, CA, United States), Collagen I (Wanlei, Shenyang, China), ALP (Abcam, Cambridge, MA, United Kingdom), Runx2, OSX (Abcam), OPN (Abcam), SNIP1 (Abcam), Smurf1 (Proteintech Group, Chicago, IL, United States), Smurf2, Smad2, Smad3, Smad4 (Cell Signaling Technology), and GAPDH (Proteintech Group). After that, the membranes were incubated with horseradish peroxidase–conjugated anti-mouse or anti-rabbit secondary antibodies (Proteintech Group) at room temperature for 1 h. Protein bands were visualized using the enhanced chemiluminescence (Millipore) and the protein levels were quantified using the ImageJ software.

Mouse ovaries were collected, and the protein was extracted with total protein extraction kits (Invent) containing phosphatase and protease inhibitor cocktails (Sigma). A BCA Protein Assay Kit (Thermo) was used to determine the protein concentration. The protein samples were separated on 8%–12% SDS-PAGE gels and then transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% non-fat dry milk for 2 h and incubated overnight at 4 °C with the following primary antibodies: phospho-cKit (Try719, CST), c-Kit (CST), phospho-AKT (Ser473, CST), AKT (CST), β-actin (CST), Bad (Abcam), Bax (Abcam), cleaved PARP1 (CST), S6K1 (CST), phospho-S6K1 (Thr389, CST), phospho-rpS6 (Ser240/244, CST), rpS6 (CST), DDX4 (Abcam), SOHLH1 (Abcam), and MSY2 (Peprotech). The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Proteintech) for 1 h at room temperature. Signals were detected with enhanced chemiluminescent substrate (Millipore).