Cytoplasmic and mitochondrial protein extraction kit

Total protein was extracted using a Whole Cell Lysis Assay (KeyGEN, China), and the cytoplasmic and mitochondrial proteins were extracted using a Cytoplasmic and Mitochondrial Protein Extraction kit (Beyotime, China). The protein concentration was determined using a BCA Protein Assay kit (KeyGEN, China). Protein samples (50 μg per lane) were separated using SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, USA) for 60 min. Subsequently, the membranes were blocked with a solution containing 5% nonfat milk/bovine serum albumin for 90 min at room temperature and then treated with specific primary antibodies at 4°C overnight. Afterwards, the membranes were incubated with a fluorescent secondary antibody for 1 h at room temperature. Then, the membranes were washed three times with TBST and scanned by a LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, USA). Densitometry was performed using ImageJ software.

Proteins were extracted from the BMSCs of each group of mice or from in vitro samples. The cytoplasmic and mitochondrial proteins were extracted using the Cytoplasmic and Mitochondrial Protein Extraction kit (Beyotime Biotechnology), according to the manufacturer’s instructions. Immunoblotting was conducted as previously described.^{93 (link)} Primary antibodies against P21 (BD Pharmingen) at 1:500, VDR (Santa Cruz Biotechnology) at 1:500, SOD1 (Santa Cruz Biotechnology) at 1:1 000, SOD2 (Santa Cruz Biotechnology) at 1:1 000, cytochrome C (Zen-Bioscience) at 1:500, AIF (Zen-Bioscience) at 1:500, cleaved caspase-3 (Zen-Bioscience) at 1:500 and Vinculin (Zen-Bioscience) at 1:2 000 were used. Immunoreactive bands were visualized with enhanced chemiluminescence (ECL) (Epizyme Biotech) and analyzed with a ChemiDoc^TM MP imaging system (Bio-Rad).