Phosphorylated serine threonine kinase p akt

Tibial bone homogenates were lysed in lysis buffer containing protease inhibitor (Roche, Mannheim, Germany) and centrifuged at 10,000× g for 10 min at 4 °C. The total protein levels were determined using a Bio-Rad protein kit (Bio-Rad, Hercules, CA, USA). The proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Immobilon-P transfer membranes (Millipore Co., Bedford, MA, USA), which were blocked with 5% bovine serum albumin prior to incubation with a specific primary antibody against BMP-2 (Abcam, Cambridge, UK), RUNX2 (Abcam), p-SMAD1/5/8 (Santa Cruz Biotechnology, Inc., Santa Cruz, TX, USA), Wnt3a (Abcam), osteocalcin (Abcam), COL-1 (Abcam), phosphorylated serine/threonine kinase (p-AKT) (Cell Signaling Technology, Danvers, MA, USA), p-ERK (Cell Signaling Technology), p38 (Cell Signaling Technology) and β-actin (Cell Signaling Technology) followed by goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP)-conjugated secondary antibody (Zymax, San Francisco, CA, USA). The antigen-antibody complexes were visualized by enhanced chemiluminescence. Densitometric analysis of the signal was performed using a C-DiGit Blot Scanner (Li-COR Inc., Lincoln, NE, USA). Relative expression was quantified using Image J (NIH, Bethesda, Rockville, MD, USA) and compared to β-actin.

Differentiated cells and tibia were homogenized in a lysis buffer containing a protease inhibitor (Roche, Mannheim, Germany) and centrifuged at refrigerated temperature (10,000 × g for 10 min at). Total protein content in the samples was quantified using a Bio-Rad DC protein assay kit. Afterward, the proteins were separated in an SDS-PAGE experiment using a 12% polyacrylamide gel. The separated proteins were transferred onto immobilon-P transfer membranes, which were blocked with 5% bovine serum albumin before incubation with specific primary antibodies against osteocalcin, COL-1, BMP-2, or RUNX2 (Abcam), SMAD5 (Santa Cruz, Texas, USA), phosphorylated serine/threonine kinase (p-AKT), p-ERK, p-p38, p-JNK, or β-actin (Cell Signaling Technology, MA, USA). The membranes were incubated with the appropriate secondary antibody, either a goat anti-rabbit IgG (H + L)-HRP conjugate or goat anti-mouse IgG (H + L)-HRP conjugate (Zymax). The immune complexes antigen–antibody were visualized using enhanced chemiluminescence. Finally, densitometric analysis of the signals was performed using a C-DiGit Blot Scanner (Li-COR, NE, USA).