Vinculin

Total proteins were extracted from GC tissues and cells using RIPA lysis buffer (Beyotime, Shanghai, China) and denatured in a 100 °C metal bath for 10 min. Based on their sizes, proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The transferred proteins were blocked using 5% skimmed milk for 1 h and incubated overnight at 4 °C with an appropriate dilution of the primary antibody. The antibodies used in this study were as follows: PTBP1 (# 32-4800, Invitrogen, Carlsbad, CA, USA), PGK1 (# ST49-07, HUABIO, Zhejiang, China), FLAG (# 14793, CST, USA), VINCULIN (# JM42-43, ZENBIO, Chengdu, China), SNAIL (# C15D3, CST, Danvers, MA, USA), β-ACTIN (# AA128, Beyotime, Shanghai, China), and Epithelial–Mesenchymal Transition (EMT) Antibody Sampler Kit (#9782, CST, Danvers, MA, USA).

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).