Bca protein concentration assay

Conditioned medium was collected after culturing MenSCs for 2 days using low-sugar medium (HyClone, USA) without fetal bovine serum. Exosomes were isolated utilizing an exosome extraction kit (Wako Pure Chemicals Industry, 293-77601) by differential centrifugation according to the manufacturer's protocol. Exosomes were verified by transmission electron microscope (TEM) (Hitachi, Japan) and a nanoparticle tracking analyzer (NTA) (ZetaView, Particle Metrix, Germany) according to a previous study [26 (link)]. Exosomes were quantified using a BCA protein concentration assay (Beyotime, China) and authenticated by western blotting using primary antibodies against TSG101 (ab83, UK), CD9 (ab223052, UK), CD63 (ab216130, UK), and calnexin (ab22595, UK). The extracted exosomes are dissolved in physiological saline for animal administration.

We extracted the total proteins of GCs using RIPA lysis buffer (Beyotime Biotechnology, Shanghai), and determined the protein concentration by BCA protein concentration assay (Beyotime Biotechnology, Shanghai). The proteins (30 µg) of each group were separated by 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with BSA for 1 h and combined with the specific primary antibody at 4 °C overnight: PI3K (Cell Signaling Technology, USA), phosphorylated (P-) AKT (Cell Signaling Technology, USA), GLUT4 (Affintiy, Jiangsu), p-GSK-3β (Cell Signaling Technology, USA), p-foxo1 (cell signaling technology, USA), and β-actin (proteintech, Wuhan). Then, the membrane was incubated with the corresponding horseradish peroxidase binding secondary antibody (proteintech, Wuhan) at 37 °C for 1.5 h. Western blots of proteins were visualized using enhanced chemiluminescence reagent (Beyotime Biotechnology, Shanghai), and quantified using image J.