Ripa buffer

Total proteins were extracted from mice gastrocnemius muscles and HUVECs using radioimmunoprecipitation (RIPA) buffer (Leagene Biotech, Beijing, China). Nuclear and cytoplasm proteins were extracted from HUVECs in lysis buffer (BestBio, Shanghai, China) following instructions from the manufacturer. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against FoxO1 (proteintech, 1:1000), Phospho-FoxO1-S256 (ABclonal, 1:1000), Akt (cell signaling, 1:1000), Phospho-Akt-S473 (cell signaling, 1:1000), β-actin (cell signaling, 1:1000), GAPDH (Unibo, 1:4000) and Lamin B1 (cell signaling, 1:1000) respectively at 4 °C overnight. On the following day, the membranes were washed and incubated with HRP-conjugated secondary antibodies (anti-rabbit secondary antibodies, cell signaling, 1:2000; anti-mouse secondary antibodies, cell signaling, 1:1000) at 4 °C for an hour. The protein-antibody interaction bands were detected by enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, USA) and visualized by a chemiluminescence detection system (Amersham Imager 600, GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

For the ALP activity assay, MC3T3-E1 cells were seeded onto titanium samples with 3 cm diameter in 6-well plates (2×10⁵ cells/well). The wells were filled with 2.5 mL 0, 2, 5, and 10% CSE in different groups. After 7 d and 14 d of incubation, the cells on the samples were rinsed with PBS and then lysed (4°C, 30 min) using radio-immunoprecipitation assay (RIPA) buffer (Leagene, China) in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF, Leagene, China). The collected lysates were then centrifugated at 12000 rpm at 4°C for 10 min. The protein concentration of the liquid supernatants was determined using a BCA protein assay kit (Keygen Biotech, China). Then, the ALP activity was assessed using an AKP assay kit (Jiancheng Bioengineering Institute, China). A total of 32 titanium samples (Ø = 30 mm) were used in this assay and equally distributed among each group. Specifically, 4 groups at 2 time points were included in this assay, and each group included four duplicate wells.