Western blot analysis

Total protein was extracted from the YYFZBJS-treated CRC cells or mouse CRC tissues using RIPA lysis buffer supplemented with a phosphatase and protease inhibitor cocktail. Protein concentration was then measured using a BCA assay kit according to manufacturer instructions. Next, 40μg of each sampled were resolved using 10% SDS-PAGE and transferred onto 0.45μm PVDF membranes (Millipore, USA). Membranes were then blocked using 5% skimmed milk in TBST for 1 h at room temperature, and then washed with TBST. The membranes were then incubated overnight (at 4°C) with antibodies against CDK1(1:1,000; Abcam), p-PI3K (1:1,000; CST), PI3K (1:1,000; CST), p-AKT (1:1,000; CST), AKT (1:1,000; CST) and β-actin (1:2000, ABclonal). The membranes were then washed and incubated with HRP-conjugated goat anti-mouse or anti-rabbit (1:1000) secondary antibodies for 2 h, at room temperature. Signal was then developed using an ECL kit.