Anti sp7 osterix rabbit polyclonal antibody

Whole cell lysates and western blot analyses were performed as described previously44 (link). The primary antibodies used were as follows: Runx2 rabbit monoclonal antibody (1:2000) (Epitomics, Burlingame CA), anti-Sp7/Osterix rabbit polyclonal antibody (1:5000) (Abcam, UK), anti-KAT3B/p300 antibody (1:2000) (Abcam), and anti-acetyl lysine antibody (1:1000) (Abcam). Membranes were incubated for 1 h at room temperature with the primary antibody in 5% milk followed by another incubation with a horseradish peroxidase-conjugated secondary antibody. The signals were detected using the Super Signal West substrate (Thermo Fisher Scientific, USA). Densitometry analyses of the western bands were performed using the Tanon Imaging software67 (link).

The protein expression of osteoblast differentiation markers was determined by western blot analysis. Briefly, cells were lysed using M-PER mammalian protein extraction reagent containing a protease inhibitor (Thermo Fisher Scientific, USA). And protein concentrations were tested with Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s specifications. Then, the lysates were separated on an 8% SDS/PAGE. After electrophoretic transfer on to nitrocellulose membranes (Thermo Fisher Scientific) and blocking with 5% milk solution, blots were incubated overnight at 4 °C with primary antibodies including anti-Runx2 rabbit monoclonal antibody (1:2000, Epitomics, CA), anti-Sp7/Osterix rabbit polyclonal antibody (1:1000, Abcam, UK), and GAPDH Rabbit Polyclonal Antibody (1:5000, Proteintech, China). Then, they were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000, Jackson, USA). The protein bands were detected and visualized by the imaging system (Tanon 5500, China) after being incubated with the SuperSignal™ West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific). Densitometry analyses of the western bands were performed using the ImageJ Imaging software.