P akt t308

Following lysis of the cells in the presence of protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA), whole-cell lysates underwent SDS-polyacrylamide gel electrophoresis, with electrophoretic transfer onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Rockford, IL, USA), and immunoblotted using anti-Mcl-1 (16225-1-AP), -Bcl-2 (12789-1-AP), -Bcl-xL (66020-1-Ig), -Bax (50599-2-Ig), -β-actin (66009-1-Ig), -ERK (16443-1-AP) (Proteintech, Rosemont, IL, USA), -p-AKT (T308; AF0832), -p-AKT (S473; AF0016) (Affinity Biosciences, Zhenjiang, Jiangsu, China), -Bim (2819), -cf-Caspase 3 (9661), -p-STAT5(Y694; 9359 S) (Cell Signaling Technologies, Danvers, MA, USA), -Bak (ab69404), -p-ERK(T202/Y204; ab4819), -AKT (ab8805), -FLT3 (ab245116) (Abcam, Cambridge, MA, USA), as previously described^{22 (link),23 (link)}. The Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA) was used to visualize immunoreactive proteins, as described by the manufacturer. Western blots were repeated, at a minimum three times, and one representative blot is displayed. The Odyssey V3.0 program (Li-Cor) was used to perform densitometry measurements, normalized to β-actin, and calculated as fold-change compared to the corresponding vehicle control.

Total protein from fracture tissues was extracted from all groups six weeks after the operation, followed by measuring concentration of protein extract with a BCA kit (Beyotime, Shanghai). Equal amounts of protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis and transferred to polyvinylidene fluoride (PVDF, Millipore, MA) membrane. The obtained PVDF membrane was incubated overnight with primary antibodies against USP1 (1:3000, Proteintech, Wuhan), BMP2 (1:1000, Affinity, Changzhou), RUNX2 (1:1000, Abclonal, Wuhan), OCN (1:500, Abclonal, Wuhan), Akt (1:1000, Affinity, Changzhou), p-Akt^T308 (1:1000, Affinity, Changzhou) and p-Akt^S473 (1:1000, Affinity, Changzhou) at 4 °C. At last, after washed by TBST (Tris-buffered saline + Tween), the membranes were incubated with secondary antibodies goat anti-rabbit IgG (Abclonal, Changzhou) and goat anti-mouse IgG (Abclonal, Wuhan) at 37 °C for 40 min. The bands were detected by enhanced chemiluminescence (ECL, Beyotime, Shanghai) detection reagent and the optical density values of the target bands were analyzed by image analysis software (Tanon, Shanghai).