Bicinchoninic acid assay

The expression of protein in cells was evaluated by Western blotting. Whole cell lysates (WCL) were prepared by extraction in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), followed by protein quantification using a bicinchoninic acid assay (Applygen, Beijing, China) and lysis in Laemmli sample buffer. A total of 30 μg of the protein sample was run on a 10% Tris‐glycine gel and transferred to nitrocellulose. Primary antibodies were added and incubated overnight at 4°C, and secondary antibodies were conjugated to horseradish peroxidase for two hours at room temperature. Blots were developed by enhanced chemiluminescence and photographed using Fujifilm Dark Box II and Image Reader LAS‐1000 Plus software (Minato‐ku, Tokyo, Japan). Primary antibodies included protein kinase B (AKT), ERK1/2, beta‐actin (antibodies at 1:2000 dilutions), and phosphorylated (p)AKT, pERK1/2 (antibodies at 1:1000 dilutions; Cell Signaling Technology). Peroxidase labeled anti‐rabbit or anti‐mouse secondary antibodies were used (Cell Signaling Technology).

Gastric cancer cells were collected and lysed using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) and quantified using a bicinchoninic acid assay (Applygen, Beijing, China). Lysates were resolved on 12% SDS-PAGE gels and electroblotted onto nitrocellulose membranes. Primary antibodies including 1B50-1 mAb, CD44 mAb (Cell Signaling Technology), ALDH1A1 mAb (Cell Signaling Technology) and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated overnight at 4°C. Then, secondary antibodies (Cell Signaling Technology) including horseradish peroxidase-labeled anti-rabbit IgG or anti-mouse IgG were added and incubated for 2 hrs at room temperature. The bands were visualized using enhanced chemoluminescence and photographed with a Fujifilm Dark Box.
The 1B50-1 mAb was provided by Zhiqian Zhang, PhD (Laboratory of Carcinogenesis and Translational Research, Department of Cell Biology, Peking University Cancer Hospital and Institute).
Antibodies used in this study are summarized in Table 1.

An ROS assay kit (Cell Biolabs, Inc., San Diego, CA, USA) was used to determine ROS levels in the rat hippocampus. Briefly, rats were sacrificed by decapitation at the end of the experiments. The brain tissues were removed rapidly, and hippocampus was dissected out on ice for subsequent experiments. Subsequently, the hippocampus was homogenized and centrifuged at 7,155 × g for 5 min at 4°C, and the total protein concentration of the supernatant was determined using a bicinchoninic acid assay (Applygen Technologies Inc., Beijing, China). Protein samples were subsequently incubated with catalyst and dichlorodihydrofluorescein solutions included in the ROS assay kit. The fluorescence was read at 480/530 nm using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).