Af5003

Primary mouse calvarial osteoblasts and bone samples from the right femurs were lysed with RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitor (Beyotime, China). The protein concentration was quantified with a BCA protein assay kit (Solarbio, China). Western blot was then performed as described previously [20 (link)]. Briefly, equal amounts of proteins from each sample were separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with primary antibodies against Runx2 (1:2000, abcam, ab236639), osteocalcin (OCN) (1:300, Beyotime, AF6300), HIF-1α (1:500, abcam, ab179483), β-catenin (1:500, abcam, ab68183) and β-actin (1:1000, Beyotime, AF5003) overnight at 4 °C. Then, the membranes were incubated with the appropriate secondary antibody (1:5000, Beyotime, A0208). The protein bands were visualized using Novex® Chemiluminescent HRP Substrate (Invitrogen, USA) and an enhanced chemiluminescence detection system (Pierce Biotechnology, USA). The band intensity was quantified with ImageJ software.

The cells were lysed with RIPA lysis buffer (P0013B, Beyotime) on ice for 30 min. The supernatants of cells were harvested after centrifugation at 12,000 g for 10 min at 4°C. A BCA protein assay kit (P0012S, Beyotime) was used to determine the total protein concentration. Protein extracts were mixed with loading buffer and boiled at 95°C for 10 min. The boiled samples were loaded onto SDS-polyacrylamide gels followed by electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with TBS-tween containing 5% skim milk, inculcated with either RUNX2 (1:1,000 dilution, S12556, Cell Signaling Technology), BMP2 (1:1,000 dilution, AF0075, Beyotime), HMOX-1 (1:1,000 dilution, AF1333, Beyotime), BID (1:1,000 dilution, AF6306, Beyotime), IL-6 (1:1,000 dilution, AF7236, Beyotime), HIF-1α (1:1,000 dilution, CSB-PA002906, CUSABIO), PRKAA2 (1:2,000 dilution, CSB-PA805325LAO1HU, CUSABIO), β-actin (1:5,000 dilution, AF5003, Beyotime), or GAPDH (1:10,000 dilution, S2118, Cell Signaling Technology) antibodies overnight at 4°C. The membranes were washed and inculcated with HRP-labeled secondary antibody (1:5,000 dilution, ab205718, Abcam). Detection was done using clarity western ECL (WBKLS0100, Millipore). The images were acquired through the e-Blot Touch Imager and semiquantitative analyses were performed by the FIJI software.

Western blot was carried out essentially as described^{30 (link)} with slight modifications. Total protein was extracted using a whole protein extraction kit (Solarbio, Beijing, China), and protein concentrations were measured using a BCA protein assay kit (Solarbio, Beijing, China). Next, total proteins were separated by SDS/PAGE electrophoresis and then transferred to a PVDF membrane (EpiZyme, Shanghai, China) at 25 °C for 1 h. Overnight incubation with the primary antibody at 4 °C was performed, and then goat anti-rabbit IgG (Beyotime, Shanghai, China) was added at 25 °C for 1 h. Finally, the proteins were detected using a developing solution (Thermo Fisher Scientific, USA). The following primary antibodies were used: CST1 (1:1000; ab307416, Abcam, British), MEK1/2 (1:1000; #9122, CST, USA), p-MEK1/2 (Ser217/221) (1:1000; #9121, CST, USA), p44/42 MAPK (Erk1/2) (1:1000; #9102, CST, USA), p-p44/42 MAPK (Erk1/2) (1:1000; #9101, CST, USA), GRIM19 (1:1000; A18071, Abclonal, China), SDHA (1:1000; A16204, Abclonal, China), UQCRC2 (1:1000; A4184, Abclonal, China), COX IV (1:1000; A6564, Abclonal, China), ATP5A1 (1:1000; A11217, Abclonal, China), VDAC1 (1:1000; A19707, Abclonal, China), MMP2 (1:1000; #4022, CST, USA), and β-actin (1:1000; AF5003, Beyotime, Shanghai, China). All experiments were repeated three times.