Western blotting

Following treatment, cells were lysed on ice for 20 min with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). A total of 20 µg protein was separated by SDS-PAGE on a 8–12% gel and electronically transferred onto a polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking at 4°C overnight with 5% no-fat milk in TBT Tween 20, the membranes were incubated with the recommended dilutions of primary antibodies against TRIAP1 (dilution, 1:800; cat. no. LS-C346398-50; LifeSpan Biosciences, Inc., Seattle, WA, USA), IPMK (dilution, 1:500; cat. no. TA337886; OriGene Technologies, Inc., Beijing, China), AMP-activated protein kinase (AMPK) (dilution, 1:1,000; cat. no. 2603), phosphorylated (p)-AMPK (dilution: 1:800; cat. no. 2535) p53 (dilution, 1:2,000; cat. no. 2524), cleaved caspase 3 (CC3) (dilution, 1:500; cat. no. 9661), Akt (dilution, 1:1,000; cat. no. 13038), p-Akt (dilution, 1:1,000; cat. no. 4060), cyclin D1 (dilution, 1:800; cat. no. 2978; all Cell Signaling Technology, Inc., Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (dilution, 1:5,000; cat. no. SC47724; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 h at 37°C. The membranes were subsequently incubated with the following secondary antibodies: Horseradish peroxidase (HRP)-conjugated anti-rabbit Immunoglobulin G (IgG; dilution, 1:10,000; cat. no. ZB2301); and the HRP-conjugated anti-mouse IgG antibody (dilution, 1:10,000; cat. no. ZB2305) both from OriGene Technologies, Inc. at room temperature for 2 h. Peroxidase-labeled bands were visualized using an enhanced chemiluminescence kit (EMD Millipore), according to the manufacturer's protocol.