Supersignal west dura chemiluminescence kit

After 24 hours of treatment with A1 or vehicle, the M17 cells were harvested in 2 x SDS-PAGE sample buffer. The samples were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. Then, the membranes were blocked in 5% milk for one hour, followed by incubation overnight at 4°C with the corresponding primary antibody. The primary antibody was anti-APP, which is specific to the APP amino terminal (Abcam, Cambridge, UK), and anti-BACE1 (Sigma Aldrich, St. Louis, MO, USA). On the subsequent day, the membranes were washed three times with TBST buffer for five minutes. The membranes were then incubated in 5% milk (v/v) supplemented with anti-rabbit IgG or anti-mouse IgG for one hour at room temperature. Following incubation with the secondary antibody, the membranes were washed three times with TBST buffer for five minutes. The blots were then visualized via incubation with a SuperSignal West Dura chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA). Images were obtained via an AlphaImager HP System (Alpha Innotech, San Leandro, CA, USA). β-actin was used as a reference control for protein loading. The bands were quantified using Fluorchem Q SA software (Alpha Innotech, Santa Clara, CA, USA).

Whole-cell lysis was performed as previously described [15 (link), 27 (link)]. The membranes were probed with anti-PTEN (1:1000, Cell Signaling, Danvers, USA) and anti-β-actin antibodies (1:2500, Santa Cruz Biotechnologies, Dallas, USA). Signals were visualized using the SuperSignal West Dura chemiluminescence kit (Thermo Fisher Scientific, Waltham, USA) and imaged on a Chemidoc gel imager (Bio-Rad, Hercules, USA). Images were quantified using the Image J software (NIH).