Ocn ab93876

Cells after indicated treatment were washed 3 times by cold PBS and dissolved with lysis buffer on ice for 20 min. After that, the cells were harvested, and after centrifugation, the supernatant containing the lysate was obtained and frozen under −80 °C. Protein qualities and concentrations were determined by the BCA assay kit (Cat. 5000001, Bio-Rad, USA). Protein samples were denatured and then separated by SDS-PAGE. The gels were then transferred into PVDF membranes (Cat. IPVH00010, Millipore, USA). After incubated with non-fat milk for 1 h, primary antibodies from Cell Signaling Technology (Danvers, USA) were used to incubate membranes under 4 °C overnight. Then the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The proteins were visualized by an ECL reagent (Thermo Fisher Scientific). Primary mouse or rabbit monoclonal antibodies (diluted at 1:1000) against GAPDH (#5174), Runx2 (#8486), ALP (#8681), KDM5B (#15327), SKP2 (#2652), Flag-tag (#14793), Myc-tag (#2276) and HA-tag (#3724) were purchased from Cell Signaling Technology (MA, USA), and OCN (ab93876) was purchased from Abcam (MA, USA). Secondary HRP-conjugated anti-mouse (#7076) or rabbit (#7074) IgG antibodies were purchased from Cell Signaling Technology (MA, USA) and used.