Supersignal west pico chemiluminescent substrate trial kit

The caput femoris samples were milled and lysed with RIPA lysis buffer (Beyotime, China) for extracting total proteins. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked for 2 h at room temperature in TBS-Tween 20 (TBST) buffer containing 5% BSA, washed with TBST three times, and incubated overnight at 4°C with 1/500 dilution of Wnt10b antibodies, LRP5 antibodies, β-catenin antibodies, Axin2 antibodies, OPG antibodies, RANKL antibodies, Dkk-1 antibodies, SOST antibodies, Runx2 antibodies, PPAR-γ antibodies, C/EBPα antibodies, FABP4 antibodies (all purchased from Santa Cruz Biotech), and GAPDH antibody (1 : 1000, BOSTER, China), respectively. After being washed with TBST, the membranes were incubated with the secondary biotin-conjugated antibody and then with anti-biotin horseradish peroxidase- (HRP-) linked antibody (1 : 1000). Protein signals were detected using SuperSignal West Pico Chemiluminescent Substrate Trial kit (Thermo Scientific) and quantified by densitometry using Quantity One software (Bio-Rad).

Edaravone injection was purchased from Jiangsu Simcere Pharmaceutical Co., Ltd. (molecular weight: 174.20). Fetal bovine serum (FBS), phosphate buffered solution (PBS), Dulbecco's modified Eagle's medium (DMEM), and trypsin (with phenol red) were purchased from Hyclone (GE Healthcare). MTT cell proliferation and cytotoxicity assay kit, Annexin V-FITC apoptosis detection kit, reactive oxygen species (ROS) assay kit, Rhodamine 123, Trizol, RIPA lysis buffer, enhanced BCA protein assay kit, and SDS-PAGE gel quick preparation kit were purchased from Beyotime. Revert aid first strand cDNA synthesis kit and Super Signal West Pico Chemiluminescent Substrate Trial kit were purchased from Thermo Fisher Scientific Inc. SYBR® Premix Ex Taq™ II was purchased from Takara Bio Inc. Bcl-2 antibodies, Bax antibodies, caspase-3 antibodies, and β-actin antibodies were purchased from Santa Cruz Biotech.