Liver tissues were cut into pieces and lysed with RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with protease inhibitors (Beijing Applygen Co., Ltd., Beijing, China) on ice for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. The protein concentration was determined by the BCA method according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, Haimen, China). Total proteins (20 μg) were separated via 12–15% SDS polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Beyotime Institute of Biotechnology). After blocking at room temperature for 2 h with 5% non-fat milk in TBS with 0.1% Tween 20, the membranes were incubated overnight at 4°C with antibodies against BECN1 (cat. no. 3495), Atg5 (cat. no. 12994), Atg7 (cat. no. 8558), and Akt (cat. no. 4691), p-Akt (Thr308) (cat. no. 13038), Raptor (cat. no. 2280), P-Raptor (Ser792) (cat. no. 2083), AMPKα (cat. no. 5832), P-AMPKα (Thr172) (cat. no. 2535), ULK1(cat. no. 8054), P-ULK1 (Ser555) (cat. no. 5869), β-actin (cat. no. 4970) and HRP-conjugated secondary antibodies (cat. no. 7074) at room temperature for 1.5 h; all antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Signals were visualized with Amersham ECL substrates, and the relative levels of protein in each group were normalized to β-actin.

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