Ecl chemiluminescence

BMSCs were collected and lysed using lysis buffer on ice for 10 min. The lysates were centrifuged at 12,000 g under 4°C for 10 min. Then, protein concentration in samples was measured by Bradford. Protein samples were separated by SDS-PAGE electrophoresis, followed by transferring to a nitrocellulose membrane (NCM). The NCM was then blocked in TBST containing 5% nonfat milk powder for 60 min, incubated with primary antibodies (RUNX2: CST, No. 8486; OPN: Boster, No. PB0589; OCN: Abcam, No. ab93876; and GAPDH: Santa, No.25778) overnight at 4°C, subsequently incubated with HRP labeled second antibody at room temperature for 1 h. The blotting bands were detected by ECL chemiluminescence (Santa Cruz, California, USA).

Briefly, cells were lysed with a lysis buffer (20 mM Tris-HCl pH 7.4, 2 mM EDTA, 25 mM NaF and 1% Triton X-100), containing protease inhibitors (Roche, Basel, Switzerland). Then the lysates were centrifuged at 4°C for 5 min at 12,000 g, and the supernatant was incubated with specific antibody and protein A/G beads overnight at 4°C. Next day, the precipitants were washed 3 times with washing buffer and eluted with sample buffer for 5 min at 95°C. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Pall, Ann Arbor, Michigan, USA). The membrane was incubated with a primary antibody overnight at 4°C, followed by incubation with secondary antibody for 1 h at room temperature. Finally, the proteins of interest were detected using ECL chemiluminescence (Santa Cruz).