Bm0627

Total proteins were extracted from the cells and separated by SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membrane. The membranes were blocked with 3% bovine serum albumin. Target proteins were detected by incubating the membranes with primary antibodies and the horseradish peroxidase-conjugated secondary antibody (Sigma) followed by the Pierce ECL Western blotting substrate (Thermo Fisher). The following primary antibodies were used for CRY1, sc-393466 (Santa Cruz); for CREB and p-CREB, #9197 and #9198 (Cell Signaling Technology); for ERK1/2 and p-ERK1/2, #4695 and #4370 (Cell Signaling Technology); for β-actin, BM0627 (Boster). Each experiment was repeated at least 3 times.

The protein expressions of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) on four different titanium specimens were analyzed by western blotting. After culturing cells (2 × 10⁵ cells per well) on different specimens in 6-well plates for 7 and 14 days, the cells were rinsed with cold PBS and protein samples were harvested by lysis in RIPA buffer. The BCA protein assay kit (Key-GEN BioTECH, Nanjing, China) was used to determine total protein concentration. Protein extract samples (20 μg) were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 hour at room temperature and then incubated with different primary antibodies against Runx2 (12556, CST, Beverly, MA, USA), OPN (ab8448, Abcam, USA), and β-actin (BM0627, Boster, Wuhan, China) overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibodies (ZB-2301, goat anti-rabbit IgG; ZSGB-BIO, Beijing, China; AP124P, goat anti-mouse IgG; Millipore, USA) for 2 hours. Finally, the membranes were visualized via ECL western blot kit (Millipore, USA). The protein expressions of Runx2 and OPN were evaluated relative to that of β-actin as the internal control. The experiments were performed in triplicate.

Protein expressions of Runx2 and OCN of cells on titanium surfaces were examined by Western blotting. After culturing in 6-well plates on different samples pre-adsorbed with 0, 50, 100 and 200 μg ml⁻¹ LDL for 3, 7 and 14 days, MC3T3-E1 cells (2 × 10⁵ cells per well) were rinsed with cold PBS and protein samples were harvested by lysis in radio-immunoprecipitation (RIPA) buffer. The concentration of extracted total proteins was determined using the BCA protein assay kit (Key-GEN BioTECH, Nanjing, China). Protein samples (20 μg) were separated by electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies against Runx2 (12556; CST, Beverly, MA, USA), OCN (ab93876; Abcam, Cambridge, Ma, USA), and β-actin (BM0627, Boster, Wuhan, China) at 4 °C overnight. On the next day, the membranes were incubated with corresponding secondary antibodies (ZB-2301, Goat anti-Rabbit IgG, ZSGB-BIO, Beijing, China; AP124P, Goat anti-Mouse IgG, Millipore, USA) at room temperature for 2 h. After that, the membranes were visualized using the ECL Western Blot Kit (Millipore, USA). The protein expressions were determined relative to that of β-actin. The gray values of protein levels were quantified using the Photoshop software.