Ecl plus detection reagent

Cells were lysed using RIPA (Sigma-Aldrich) buffer containing a mixture of protease inhibitors. Protein concentrations in cells lysates were determined using a BCA (Thermo Fisher Scientific) kit. Protein from lysed cells (50 μg) was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, and this was followed by blocking for 2 h. Next, membranes were incubated overnight with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Antibodies for immunoblotting, including β-actin (ab8226, 42 kDa), Runx2 (ab92336, 57 kDa), OCN (ab2360486, 11 kDa), and β-Catenin (ab16051, 94 kDa) were purchased from ABCAm (all 1: 1,000 dilutions). The protein bands were visualized with ECL Plus Detection Reagent (Applygen, Beijing, China).

Proteins (50 μg) from lysed cells were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, then blocked for 2 h. Next, the membranes were incubated overnight with primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The protein bands were visualized using ECL Plus Detection Reagent (Applygen, Beijing, China).

Proteins from lysed cells (50 μg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, and this was followed by blocking for 2 h. Next, membranes were incubated overnight with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized with ECL Plus Detection Reagent (Applygen, Beijing, China).