Total protein extract kit

Twenty 6-week old nude mice (nu/nu) were randomized into a control group (n = 10) and a LOX inhibition group (n=10). The mice were fed a standard diet every day and housed under conditions with a light-dark cycle of 12 h/12 h, temperature of 22°C ± 2°C, and humidity of 55% ± 5% for 1 week of acclimatization before the experiments. In the LOX inhibition group, mice were given daily 0.2 mL intraperitoneal injections of the LOX inhibitor, BAPN (Sigma, St. Louis, MO, USA) at a dose of 100 mg/kg. The same volume of sterile PBS was administered via intraperitoneal injections to the control group every day. After 2 weeks, 1×10⁷ SGC-7901 gastric cancer cells/mouse were inoculated subcutaneously into the backs of the two groups of mice to create the gastric cancer-bearing nude mouse model. BAPN or PBS intraperitoneal injections were still administered. After 4 weeks from inoculation of tumor cells, and when model construction was successful, mice from the two groups were anesthetized using ether and euthanized by cervical dislocation. In situ tumors were extracted, and total protein was extracted from the mouse xenograft tumors following the manufacturer's instructions in the total protein extract kit (KeyGEN BioTECH Jiangsu, P.R. China). The BCA assay was used for protein quantitation.

Distal colonic samples (2–3.5 cm proximal to the anus) were ground and lysed using a total protein extract kit (Keygen). Each sample (30 μg protein) was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Wang et al., 2016 (link)). The membrane was first incubated with primary antibodies against Caspase 3, PCNA, mTORC1, and GCN2 (Cell Signaling) overnight at 4°C and then incubated with the secondary antibody for 2 h at 25°C. The results were visualized via chemiluminescence with EZ-ECL (Biological Industries).