Anti runx2 ab23981

WAT or cells were homogenized in buffer with protease and phosphatase inhibitors. Total protein was extracted with TRlzol Reagent (Sigma). Protein content was determined with a protein assay kit, and samples were then aliquoted and stored at −80 °C. The protein was separated by SDS-PAGE and transferred to nitrocellulose membranes, followed by blocking for 2 h and incubation for 12 h at 4 °C with primary antibodies. Anti-Insulin Receptor β (#3025), anti-Phospho-insulin receptor β (#3024), Phospho-Akt (Ser473) (#4060), and anti-Akt (#4691) antibodies were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA); anti-phospho-PPARγ (ser273) (bs-4888R) was purchased from Bioss Inc. (Woburn, MA, USA); and anti-PPAR gamma (ab59256), anti-Osteocalcin (ab93876) and anti-Runx2 (ab23981) were obtained from Abcam (Cambridge, UK). The membrane was then incubated with the secondary antibodies for 1 h at room temperature. The protein bands were visualized with an enhanced chemiluminiscence (ECL) detection system. The band intensity was quantified with Fusion FX (Vliber Lourmat, Australia) software and all quantitative analyses were normalized to β-actin or GAPDH.