2.2. Western Blot Analysis

Western blot assays were performed as previously described [11]. Briefly, total proteins containing the target proteins TRPP2, AMPK, p-AMPK, ACC, p-ACC, PERK, p-PERK, eIF2α, and p-eIF2α were extracted from HN-4 cells with a detergent extraction buffer. Total protein extracts (30 μg) were loaded into each lane of a 10% sodium dodecyl sulfate polyacrylamide gel, separated by gel electrophoresis, and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore). Following transfer, membranes containing the target proteins were incubated in Tris-buffered saline solution supplemented with 10% nonfat milk for 1 h at room temperature to block nonspecific binding sites. Subsequently, for immunoblots, membranes were first incubated with the primary antibodies against TRPP2 (cat. no. sc-25749), PERK (cat. no. sc-13073), p-PERK (cat. no. sc-32577), eIF2α (cat. no. sc-11386), p-eIF2α (cat. no. sc-101670; all from Santa Cruz Biotechnology, Inc.), AMPKα (cat. no. 2532S), p-AMPK (Thr172 in the α subunit) (cat. no. 40H9), ACC (cat. no. C83B10), and p-ACC (Ser79) (cat. no. D7D11; all from Cell Signaling Technology, Inc.) at a 1 : 200 dilution overnight at 4°C and then with the corresponding secondary antibodies for 1 h at room temperature. The immunosignals were detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.). The optical density (OD) of each protein band was analyzed using the ImageJ software (National Institutes of Health) by calculating the integrated OD values. The results are expressed as the relative OD. All assays were performed in triplicate.