Protein extraction kit

The expression levels of APN, APR1, APPL1, AMPK, pAMPK, FOXO1, and pFOXO1 in the brain and primary neurons were analyzed using Western blot analysis. Proteins from the brain tissues around the infarct area and primary neurons were collected using a protein extraction kit (Epizyme). Bicinchoninic acid assay was used to detect the concentration of proteins. An equal number of proteins in different groups were separated by Tris–glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Consequently, the membranes were blocked with 5% nonfat milk in PBST at room temperature for 1 h and incubated with primary antibodies (anti-APN, Abcam; anti-APR1, Proteintech; anti-APPL1, Proteintech; anti-AMPK, Abcam; anti-pAMPK, Abcam; anti-FOXO1A, Abcam; anti-pFOXO1A, Abcam; and anti-ACTB antibody, Sigma) at 4°C overnight. On the following day, the membranes were incubated with corresponding HRP-conjugated secondary antibody (Abcam) at room temperature for 1 h. The protein band was scanned on Amersham Imager 600 using an enhanced chemiluminescence kit (Sangon Biotech (Shanghai) Co.), and the relative amounts of proteins were analyzed using ImageJ software.